A method for reducing the stability of amorphous calcium phosphate

Abstract

This invention discloses a method for reducing the stability of amorphous calcium phosphate. The method involves adding an additive solution to an ACP solution; the additive is a calcium- and phosphorus-free inorganic salt that does not react with calcium ions and orthophosphate ions to form a precipitation reaction. This invention, by adding an inorganic salt after the formation of amorphous calcium phosphate, primarily increases the ionic strength of the solution, reduces calcium and phosphorus activity, promotes the dissolution of amorphous calcium phosphate, thereby altering its structure, significantly reducing its stability, and accelerating the formation of hydroxyapatite. Furthermore, this method uses low-cost raw materials, contributing to cost reduction.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic mineralization materials and hard tissue repair technology, specifically relating to a method for reducing the stability of amorphous calcium phosphate. Background Technology

[0002] Amorphous calcium phosphate (ACP) is considered a precursor material for hard tissue biomineralization and the synthesis of calcium phosphate materials. The stability of ACP generally refers to its stability in aqueous solution, which can be measured by the time from ACP formation to the start of its transformation into calcium phosphate crystals, i.e., lifetime or induction period. The bidirectional regulation of ACP stability is fundamental to achieving multi-level structures in calcium phosphate materials. For example, the nanoscale structural unit of bone is mineralized collagen. In vitro studies have found that during the formation of mineralized collagen, ACP is initially stabilized, transported to the collagen as a calcium and phosphate storage capsule, and subsequently destabilized, transforming into bone apatite to form mineralized collagen. If ACP cannot be actively destabilized, it must be waited for either natural destabilization or point-dependent destabilization. The former reduces the mineralization efficiency of ACP, while the latter increases the difficulty of the process.

[0003] Current techniques for destabilizing ACP include increasing temperature, decreasing pH, adding calcium salts or orthophosphates, and organic matter. Increasing temperature reduces ACP stability. Decreasing pH also reduces ACP stability. Increasing the concentration of calcium salts or orthophosphates reduces ACP stability because calcium and orthophosphate are the main components of calcium phosphate; increasing their concentrations increases calcium-phosphorus activity, thus reducing ACP stability. Adding organic matter reduces ACP stability; currently, amino acids, peptides, proteins, and polyelectrolytes can all reduce ACP stability due to the aggregation of functional groups (calcium-phosphorus ions) or the template effect of macromolecules.

[0004] Existing technologies suffer from high costs and limited application environments, while calcium- and phosphorus-free inorganic salts (hereinafter referred to as inorganic salts) can partially solve these problems. The total production and waste disposal costs of temperature control equipment, peptides, polyelectrolytes, amino acids, calcium salts, and phosphate salts are higher than those of inorganic salts such as sodium chloride and ammonium chloride. In applications, biomimetic mineralization and hard tissue repair often involve heat-sensitive materials such as proteins or occur within the human body, where temperatures are typically constant and should not be increased. Amino acid-based materials and polyelectrolytes contain a large number of carboxyl or amino functional groups, exhibiting stronger pH buffering capacity than sodium chloride and ammonium chloride. Increasing their concentration can significantly alter the solution pH, making them difficult to use in scenarios requiring constant pH solutions. Calcium and phosphate salts can precipitate with common or functional ions such as carbonate, magnesium, and strontium ions. In some scenarios, increasing the calcium and phosphorus concentration can introduce impurities. In contrast, inorganic salts such as sodium chloride and ammonium chloride rarely form precipitates with non-heavy metal ions, and ammonium ions are easily removed.

[0005] However, currently reported inorganic salts only possess the function of improving ACP stability; these include sodium, chloride, magnesium, carbonate, zinc, and iron ions. Unlike calcium and orthophosphate salts, which increase calcium and phosphorus activity, inorganic salts can increase ionic strength and decrease the activity coefficient, thereby reducing calcium and phosphorus activity and thus improving ACP stability. Furthermore, the stabilizing effect of inorganic salts on ACP is also attributed to their adsorption on the ACP surface or their incorporation into the ACP interior.

[0006] No technology has yet been developed to reduce the stability of ACP based on inorganic salts such as sodium chloride and ammonium chloride. Summary of the Invention

[0007] The purpose of this invention is to provide a method for reducing the stability of amorphous calcium phosphate (ACP). The method of this invention can reduce the stability of ACP based on inorganic salts, where the inorganic salts are sodium chloride, ammonium chloride, and other inorganic salts that do not form slightly soluble or sparingly soluble precipitates with calcium phosphate at room temperature.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for reducing the stability of amorphous calcium phosphate includes the following steps:

[0010] Add an additive solution to the ACP solution; the additive is a calcium- and phosphorus-free inorganic salt that does not react with calcium ions and orthophosphate ions (phosphate, hydrogen phosphate, dihydrogen phosphate) ions to form a precipitation reaction.

[0011] Preferably, the molar ratio of total calcium ions to total orthophosphate ions in the ACP solution is 0.5 to 2:1. More preferably, the molar ratio of total calcium ions to total orthophosphate ions in the ACP solution is 1:1.

[0012] Preferably, the total calcium ion concentration in the ACP solution is 1 mM to 0.5 M. More preferably, the total calcium ion concentration in the ACP solution is 1 mM to 10 mM.

[0013] Preferably, the ACP solution is obtained by mixing a soluble calcium salt solution and a soluble orthophosphate solution.

[0014] More preferably, the concentration of the soluble calcium salt in the soluble calcium salt solution is 2 mM to 1 M; more preferably, the concentration of the soluble calcium salt in the soluble calcium salt solution is 2 to 20 mM.

[0015] More preferably, the concentration of soluble orthophosphate in the soluble orthophosphate solution is 2 mM to 1 M; more preferably, the concentration of soluble orthophosphate in the soluble orthophosphate solution is 2 to 20 mM.

[0016] More preferably, the molar ratio of calcium ions in the soluble calcium salt solution to orthophosphate ions in the soluble orthophosphate solution is 0.5 to 2:1.

[0017] More preferably, the soluble calcium salt is at least one of calcium chloride and calcium nitrate;

[0018] More preferably, the pH of the soluble orthophosphate solution is 7-10; adjusted using hydrochloric acid or sodium hydroxide.

[0019] More preferably, the soluble orthophosphate is at least one selected from trisodium phosphate, disodium hydrogen phosphate solution, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, triammonium phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate.

[0020] More preferably, other compounds that improve stability, such as sodium chloride and amino acids, are added during the preparation of the ACP solution.

[0021] More preferably, the soluble calcium salt solution and the soluble orthophosphate solution are mixed and stirred for 10 seconds to 10 minutes before the additive solution is added.

[0022] Preferably, the concentration of the additive in the additive solution is 2 mM to 5 M; more preferably, the concentration of the additive in the additive solution is 100 mM to 1 M.

[0023] Preferably, the additive is a salt formed by at least one of lithium ions, sodium ions, potassium ions, and ammonium ions and at least one of chloride ions and nitrate ions.

[0024] More preferably, the additive is one or more of sodium chloride, potassium chloride, lithium chloride, ammonium chloride, and sodium nitrate.

[0025] Preferably, the molar ratio of the additive in the additive solution to the total calcium ions in the ACP solution is 1–40:1. More preferably, the molar ratio of the additive in the additive solution to the total calcium ions in the ACP solution is 2–32:1.

[0026] Preferably, the volume ratio of the additive solution to the ACP solution is 0.01 to 0.1:1.

[0027] Preferably, the ACP solution and the additive solution (and the mixed solution) are in a stirring state; the stirring speed is 20 to 800 rpm.

[0028] This invention reduces the stability of ACP by adding inorganic salts after ACP formation. These inorganic salts react with calcium ions or orthophosphate ions to form salts that are readily soluble or extremely soluble in water at room temperature, including but not limited to salts formed by any combination of lithium, sodium, potassium, ammonium, chloride, and nitrate ions. The inventors believe that these inorganic salts reduce calcium and phosphorus activity or increase the osmotic pressure of the solution. Adding these inorganic salts after ACP formation reduces the calcium and phosphorus activity in the solution, disrupting the dissolution-reprecipitation equilibrium of ACP in the suspension, inducing ACP dissolution, and thus altering the ACP structure. For example, it may reduce the particle size of ACP, thereby decreasing its stability, or increase the osmotic pressure of the solution, promoting the removal of adsorbed water from ACP and accelerating its transformation into calcium phosphate crystals with very low adsorbed water content. Since these inorganic salts do not form sparingly soluble or slightly soluble precipitates with calcium and phosphorus, they do not increase the stability of ACP by encapsulating it and isolating it from the solution.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) Existing technologies have not yet achieved the reduction of ACP stability using calcium- and phosphorus-free inorganic salts. This is because they use salts such as magnesium ions and fluoride ions to form slightly soluble or sparingly soluble precipitates with calcium and phosphorus at room temperature to improve ACP stability, or they directly mix salts such as NaCl with calcium and phosphorus to form salts that are easily soluble in water at room temperature to improve ACP stability, i.e., adding salts such as NaCl before ACP formation. However, this invention discovers that reducing ACP stability is achieved simply by changing the addition time of salts such as NaCl. The process is simple. It does not use expensive amino acids, peptides, polyelectrolytes, or temperature control equipment, resulting in lower costs and better or no reduction in effectiveness. It does not use reagents such as acids, alkalis, calcium, or phosphorus that significantly alter the solution pH or easily form insoluble impurities. Furthermore, salts such as NaCl can inhibit the formation of insoluble impurities through the salt effect, broadening its application range.

[0031] (2) This invention demonstrates that combining ACP, addition time, and salt type produces effects unpredictable by existing theories, which is significant for refining non-classical crystallization theories and understanding biomineralization. Currently, it has been found that increasing calcium and phosphorus concentration decreases ACP stability, attributed to the increased calcium and phosphorus activity of calcium and phosphate salts. Inorganic salts have been found to only increase ACP stability, attributed to their reduced calcium and phosphorus activity, adsorption on the ACP surface, or incorporation into the ACP interior. Organic compounds such as amino acids, peptides, proteins, and polyelectrolytes have been found to decrease ACP stability and promote ACP-mediated remineralization. The former is attributed to the aggregation of calcium and phosphorus by organic functional groups, increasing local calcium and phosphorus concentration, or the template effect of macromolecular conformation. The latter is attributed to the organic compounds promoting the binding (deposition) between ACP and collagen and the entry of ACP into collagen pores, rather than subsequent ACP instability and crystallization. Based on existing theories, it can be predicted that adding salts such as NaCl will decrease calcium and phosphorus activity, thereby increasing ACP stability. The results of this invention cannot be predicted, i.e., adding inorganic salts such as NaCl after ACP formation will decrease ACP stability. Attached Figure Description

[0032] Figure 1 The graph shows the effect of the order of NaCl addition on the stability of ACP in Example 1.

[0033] Figure 2 The graph shows the effect of the order of adding different concentrations of NaCl on the stability of ACP in Example 1.

[0034] Figure 3 The graph shows the effect of the order of addition of the inorganic salts (which form slightly soluble or sparingly soluble salts with calcium and phosphorus) described in Example 2 on the stability of ACP.

[0035] Figure 4 The graph shows the effect of the order of addition of the inorganic salts (which form water-soluble salts with calcium and phosphorus) described in Example 3 on the stability of ACP.

[0036] Figure 5 This is a graph showing the effect of NaCl on the stability of the stabilized ACP in Example 4.

[0037] Figure 6 This is a graph showing the effect of the order of amino acid addition described in Example 5 on the stability of ACP.

[0038] Figure 7 This is a morphological diagram of the mineral obtained in Example 6.

[0039] Figure 8 This is a graph showing the effect of the minerals obtained in Example 6 on the alkaline phosphatase activity of mesenchymal stem cells. Detailed Implementation

[0040] The present invention will be specifically described below with reference to the embodiments, but the implementation and protection scope of the present invention are not limited to the following embodiments.

[0041] Example 1

[0042] This embodiment uses NaCl as an example to demonstrate the effectiveness of this technical solution. The specific method is as follows:

[0043] A calcium chloride solution (C = 10 mM, V = 30 mL) was poured into a disodium hydrogen phosphate solution (C = 10 mM, pH = 7.4, V = 30 mL) at a flow rate of 15–30 mL / s to obtain a suspension containing ACP, referred to as the first suspension (C). ACP =0.05~0.2mg / ml, pH=6.8~7.0), with the stability of ACP synthesized by this method as a control.

[0044] A calcium chloride solution (C = 10 mM, V = 30 mL) was poured into a disodium hydrogen phosphate solution (C = 10 mM, pH = 7.4, V = 30 mL) at a speed of 15–30 mL / s to obtain a suspension containing ACP. After stirring for 25–30 s, a NaCl solution (C = 550 mM, V = 6 mL or C = 0 mM, V = 6 mL) was poured into the suspension at a speed of 5–10 mL / s to obtain a second suspension (C = 10 mM, V = 30 mL / s). 总钙 =C 总磷 =4.5mM, C NaCl =50mM), to reduce the stability of ACP.

[0045] A NaCl-calcium chloride mixture was obtained by mixing calcium chloride solution and NaCl dry powder. 钙 =10mM, C NaCl =100mM, V=30mL), the NaCl-calcium chloride mixture was poured into the disodium hydrogen phosphate solution (C=10mM, pH=7.4, V=30mL) to obtain a suspension containing ACP, which is called the third suspension (C=100mM, V=30mL). 总钙 =C 总磷 =5mM, C NaCl =50mM), to increase the stability of ACP, and use this as a reference to illustrate the importance of the order of NaCl addition.

[0046] All reagents used were at least analytical grade, and the water was ultrapure. The solution and environment were maintained at 25℃±1℃. The solution was stirred throughout the process (200~800rpm), and the pH of the solution was adjusted using hydrochloric acid or fresh sodium hydroxide solution. All beakers and magnetic surfaces used were made of polytetrafluoroethylene (PTFE), and after mineralization and before cleaning, they were soaked in acetic acid solution (pH 1~3, C=10~30vol%) for 1~10min to remove the deposited calcium phosphate and restore the hydrophobic surface.

[0047] To characterize the stability of ACP in situ, the pH evolution before and after mixing with orthophosphate solutions was monitored.

[0048] To characterize the stability of ACP in situ, the first, second, and third suspensions were filtered and then freeze-dried or vacuum-dried to obtain ACP dry powder, which was then characterized by XRD and IR.

[0049] To further verify the effectiveness of this technique, the concentration of NaCl was changed (C NaCl =1~300mM, specifically 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250mM), while keeping the phosphorus and calcium concentrations constant, and repeat the above steps.

[0050] Figure 1 In the diagram, A is a schematic diagram of the method described in Example 1, where the addition of additives before the formation of ACP is referred to as premix, and the addition of additives after the formation of ACP is referred to as postmix. Figure 1 B in the figure represents the stability of ACP using a pH meter. The duration of the first plateau (between pH 6.8 and 7.3) in the figure can characterize the stability of ACP. Therefore, it can be seen that adding NaCl before the formation of ACP can improve the stability of ACP, while adding NaCl after the formation of ACP can reduce the stability of ACP. Figure 1 C in the figure represents the stability of ACP by XRD and IR. The results show that adding NaCl before ACP formation results in the absence of pure hydroxyapatite (HA) after 30 minutes, while adding NaCl after ACP formation results in the formation of pure HA within 10 minutes. This indicates that adding NaCl before ACP formation improves the stability of ACP, while adding NaCl after ACP formation decreases the stability of ACP. Figure 1 It can be seen that adding NaCl after ACP formation reduces the stability of ACP, while adding NaCl before ACP formation increases the stability of ACP.

[0051] Figure 2 The 'A' in the diagram illustrates the method for extracting ACP survival time based on pH data. Figure 2 In Example 1, the ACP survival time in the Control group (B) was 13.4 ± 1.5 min. This was obtained by first measuring the ACP survival time at a given NaCl concentration (1–250 mM) and then dividing that time by 13.4 min. Figure 2 The x-coordinate in C is Figure 2 The C in the figure indicates that when the NaCl concentration is less than 180 mM, adding NaCl after the formation of ACP can reduce the stability of ACP.

[0052] Example 2

[0053] This embodiment demonstrates the uniqueness of the present invention, and the specific method is as follows:

[0054] This embodiment is basically the same as Embodiment 1, except that: Embodiment 1 uses NaCl, while this embodiment uses sodium carbonate, sodium sulfate and sodium fluoride in sequence.

[0055] Figure 3 This indicates that adding sodium carbonate, ammonium sulfate, and sodium fluoride after the formation of ACP will not accelerate the pH drop, i.e., it will not reduce the stability of ACP. Combined with Example 1, it can be seen that the combination of forming room-temperature soluble salts with calcium and phosphorus such as NaCl and adding inorganic salts after the formation of ACP is the unique feature of this invention.

[0056] Example 3

[0057] This example demonstrates the universality of the present invention, and the specific method is as follows:

[0058] This embodiment is basically the same as Embodiment 1, except that: only NaCl was used in Embodiment 1, while LiCl, KCl, NH4Cl and NaNO3 were used in sequence in this embodiment; only disodium hydrogen phosphate was used in Embodiment 1, while disodium hydrogen phosphate, dipotassium hydrogen phosphate, diammonium hydrogen phosphate, diammonium dihydrogen phosphate and triammonium phosphate were used in sequence in this embodiment.

[0059] Figure 4 The pH evolution of this embodiment shows that adding the inorganic salt or orthophosphate used in this embodiment after the formation of ACP will reduce the survival time of ACP, that is, adding the inorganic salt used in this embodiment after the formation of ACP can reduce the stability of ACP.

[0060] Example 4

[0061] This embodiment further demonstrates the universality of the present invention, and the specific method is as follows:

[0062] This embodiment is basically the same as Embodiment 1, except that NaCl is added before and after the formation of ACP.

[0063] Figure 5 This is a pH evolution diagram of this embodiment. Adding NaCl only before the formation of ACP can increase the stability of ACP, while adding NaCl before and after the formation of ACP can reduce the stability of ACP. Therefore, NaCl can destabilize ACP that is stabilized by NaCl.

[0064] Example 5

[0065] This example demonstrates the advantages of the present invention, and the specific method is as follows:

[0066] This embodiment is basically the same as Embodiment 1, except that NaCl is replaced with L-Arg, L-Asp, and Gly. Taking L-Arg as an example: NaCl dry powder, NaCl solution (C = 550mM, V = 6mL), and NaCl-calcium chloride solution (C... 钙 =10mM, C NaCl =100mM, V=30mL) was changed to L-Arg dry powder, L-Arg solution (C=550mM, pH=7.4, V=6mL) and L-Arg-calcium chloride solution (C=100mM, V=30mL). 钙 =10mM, C L-Arg =100mM, pH=7.4, V=30mL), therefore the second turbid solution (C) of this embodiment can be obtained. 总钙 =C 总磷 =4.5mM, C L-Arg =50mM) and the third turbid liquid (C 总钙 =C 总磷 =5mM, C L-Arg =50mM).

[0067] Figure 6 In this embodiment, A represents the effect of L-Arg on the stability of ACP. Figure 6 B in the equation represents the effect of L-Asp on ACP stability. Both equations indicate that adding L-Arg or L-Asp before ACP formation prolongs ACP survival time, while adding L-Arg or L-Asp after ACP formation shortens ACP survival time. Figure 6 C data processing methods and Figure 2 The C in them is the same. Figure 6 The C value indicates that the stability of ACP can be reduced by adjusting the order of addition of L-Arg, L-Asp, and Gly within the range of 1–100 mM. Figure 6 and Figure 2 The comparison shows that amino acids can reduce the stability of ACP by up to 40%, while NaCl can reduce the stability of ACP by up to 20%. NaCl is more effective than amino acids in reducing the stability of ACP.

[0068] Example 6

[0069] This example further demonstrates the advantages of the present invention, and the specific method is as follows:

[0070] In this embodiment, ACP with different stabilities from Examples 1-5 was prepared into dry powder and characterized by SEM.

[0071] The ACP from Examples 1-5 was made into a dry powder, sterilized with ultraviolet light, and then an extract or suspension was prepared using a complete culture medium. Its ability to induce osteogenic differentiation of mesenchymal stem cells was evaluated based on ALP staining (ACP-2min, 45S5 bioglass, hydrothermal synthesized hydroxyapatite HA and β-tricalcium phosphate TCP were used as controls).

[0072] The steps for hydrothermal synthesis of hydroxyapatite are as follows: Calcium nitrate solution (C = 0.5 M, V = 12.5 mL) is poured into a stirred sodium phosphate solution (C = 0.1 M, V = 37.5 mL) using a peristaltic pump (0.5 mL / min). The pH is adjusted to 12 with sodium hydroxide. The mixture is then hydrothermally reacted at 150 °C for 12 h in a reaction vessel. The precipitate is collected by centrifugation, washed with deionized water and anhydrous ethanol, dried at 60 °C, and ground before use.

[0073] Figure 7 The image shows the morphology of HA. By adding NaCl or amino acids after the formation of ACP to accelerate the mineralization of ACP, it can be seen from the comparison that the HA formed after NaCl reduces the stability of ACP is still nano-needle-shaped, which has the same structure as the HA obtained without any additives and with the addition of amino acids. This shows that the present invention does not cause significant changes in the structure of HA.

[0074] Figure 8 The staining results showed that the ALP activity of mesenchymal stem cells stimulated by the obtained minerals were similar across all experimental groups. This indicates that adding additives after ACP formation does not significantly reduce the osteogenic activity or cell compatibility of the minerals.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method of reducing the stability of amorphous calcium phosphate, characterized by, Includes the following steps Add an additive solution to the ACP solution; the additive is a calcium- and phosphorus-free inorganic salt that does not react with calcium ions and orthophosphate ions to form a precipitation reaction; the concentration of the additive in the additive solution is 2 mM to 550 mM. The molar ratio of total calcium ions to total orthophosphate ions in the ACP solution is 0.5~2:1; the ACP solution is obtained by mixing a soluble calcium salt solution and a soluble orthophosphate solution. The soluble calcium salt solution and the soluble orthophosphate solution are mixed and stirred for 10 seconds to 10 minutes before the additive solution is added. The additive is a salt formed from at least one of lithium ions, sodium ions, potassium ions, and ammonium ions and at least one of chloride ions and nitrate ions.

2. The method for reducing the stability of amorphous calcium phosphate according to claim 1, characterized in that, The concentration of the soluble calcium salt in the soluble calcium salt solution is 2 mM to 1 M; The concentration of soluble orthophosphate in the soluble orthophosphate solution is 2 mM to 1 M.

3. The method for reducing the stability of amorphous calcium phosphate according to claim 1, characterized in that, The soluble calcium salt is at least one of calcium chloride and calcium nitrate; The pH of the soluble orthophosphate solution is 7-10; The soluble orthophosphate is at least one of trisodium phosphate, disodium hydrogen phosphate solution, sodium dihydrogen phosphate, tripotassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, triammonium phosphate, diammonium hydrogen phosphate, and ammonium dihydrogen phosphate.

4. The method for reducing the stability of amorphous calcium phosphate according to claim 1, characterized in that, The molar ratio of the additive in the additive solution to the total calcium ions in the ACP solution is 1~40:

1.

5. The method for reducing the stability of amorphous calcium phosphate according to claim 1, characterized in that, The volume ratio of the additive solution to the ACP solution is 0.01~0.1:

1.

6. The method for reducing the stability of amorphous calcium phosphate according to claim 1, characterized in that, The ACP solution and the additive solution are stirred; the stirring speed is 20~800 rpm.

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