Tricalcium phosphate bone cement doped with hydroxyapatite and preparation method thereof

Abstract

The present invention discloses a tricalcium phosphate bone cement doped with hydroxyapatite and a preparation method thereof, wherein the bone cement is composed of a solid phase component and a liquid phase component, wherein the solid phase component is α-tricalcium phosphate and β-tricalcium phosphate, and the addition of Mg and β-tricalcium phosphate is carried out. 2+ 、Zn 2+ 、Sr 2+ 、Cu 2+ and Si 4+ The liquid phase of this inorganic ion-doped hydroxyapatite is a mixed aqueous solution of citric acid monohydrate and disodium hydrogen phosphate dodecahydrate. Five types of doped hydroxyapatite are selected based on the trace elements present in human bone and added to bone cement. In clinical practice, the inorganic ions released as the cement degrades can significantly promote cell proliferation and new bone formation, shortening treatment time.

Description

Technical Field

[0001] The invention relates to tricalcium phosphate bone cement doped with hydroxyapatite with multiple inorganic ions and a preparation method thereof, belonging to the technical field of biomedical materials. Background Art

[0002] The solid phase of calcium phosphate bone cement is composed of one or more calcium phosphate powders. These powders form a slurry with a certain viscosity after mixing with the liquid phase. After filling the bone defect, it can self-solidify and harden in situ. It has excellent properties such as bioactivity, osteoconductivity, injectability and formability, and has great application potential in the field of bone defect repair.

[0003] Common solid phase components of calcium phosphate bone cement include dibasic calcium phosphate dihydrate (DCPD), α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP), tetracalcium phosphate (TTCP) and hydroxyapatite (HA), etc. The solid phase system is often composed of a variety of calcium phosphate powders.

[0004] It is now recognized that normal human bone metabolism requires the participation of multiple trace elements: magnesium regulates bone mineralization and bone metabolism, promoting bone tissue growth; zinc has excellent osteoinductivity and trace zinc also has certain antibacterial properties; strontium enhances osteoblast activity and inhibits osteoclast bone resorption; copper induces angiogenesis and promotes the differentiation of bone marrow mesenchymal stem cells; silicon can induce angiogenesis and regulate the biomineralization process. Incorporating inorganic ions contained in natural human bone into bone cement can promote new bone formation.

[0005] In recent years, the research on the introduction of inorganic ions into bone repair materials has mainly focused on the doping of hydroxyapatite. 2+ 、Zn 2+ 、Sr 2+ and Cu 2+ etc., can replace Ca in hydroxyapatite crystals 2 +, which does not affect the biocompatibility of the material. However, current research focuses on single-ion doping, such as CN202210201997.1, which discloses a one-step synthesis of iron-doped hydroxyapatite and its preparation method and application; there are also some studies on dual-ion doping, such as CN202111075908.5, which involves a collagen-templated biomimetic mineralized zinc-strontium-doped hydroxyapatite and its preparation method; research on multiple ion doping is rare. Patent CN201610127407.X introduces nine elements, Si, Sr, F, Mg, Zn, Na, K, Al, and C, into hydroxyapatite to prepare a multi-ion co-doped hydroxyapatite powder material. In addition, there are few studies on the use of doped hydroxyapatite as a solid phase component of bone cement. For example, CN201611036752.9 involves a composite bone cement with bioactivity and antibacterial properties, as well as its preparation method and application. The solid phase component is composed of a mixture of calcium sulfate hemihydrate and silver strontium-doped hydroxyapatite; Dai J, Fu Y, Chen D, et al. A novel and injectable strontium-containing hydroxyapatite bone cement for bone substitution: A systematic evaluation [J]. Materials Science & Engineering C: Materials for Biological Applications, 2021, 124: 112052. A novel injectable bone cement was prepared using α-tricalcium phosphate and strontium-doped hydroxyapatite as the solid phase. The existing research has the following deficiencies: (1) There are few studies on the application of doped hydroxyapatite in bone cement; (2) The types of doped ions in bone cement are relatively few, and doping with multiple ions is mainly achieved by simultaneously introducing multiple ion sources into pure hydroxyapatite. Summary of the Invention

[0006] Purpose of the invention: In response to the above-mentioned problems, the first purpose of the present invention is to provide a tricalcium phosphate bone cement with hydroxyapatite doped with multiple inorganic ions. The second purpose of the present invention is to provide a method for preparing the tricalcium phosphate bone cement with hydroxyapatite doped with multiple inorganic ions, introducing multiple inorganic ions with osteogenesis-promoting properties into the bone cement to shorten the bone defect repair time; the use of hydroxyapatite doped with multiple inorganic ions is beneficial to maintaining a slightly alkaline environment at the bone defect site and promoting bone defect repair.

[0007] Technical solution: The present invention discloses a tricalcium phosphate bone cement with hydroxyapatite doped with multiple inorganic ions, characterized in that the tricalcium phosphate bone cement comprises a mixture of a solid phase component and a liquid phase component; wherein the solid phase component comprises α-tricalcium phosphate (α-TCP), β-tricalcium phosphate (β-TCP) and hydroxyapatite doped with multiple inorganic ions, the liquid phase component comprises a mixed aqueous solution of citric acid monohydrate and disodium hydrogen phosphate dodecahydrate, and the hydroxyapatite doped with multiple inorganic ions comprises Mg doped 2+ Hydroxyapatite, doped with Zn 2+ Hydroxyapatite, doped with Sr 2+ Hydroxyapatite, doped Cu 2+ Hydroxyapatite and doped Si 4+ of hydroxyapatite.

[0008] Furthermore, the mass percentage of the α-tricalcium phosphate, β-tricalcium phosphate and hydroxyapatite doped with various inorganic ions is 6-3:3-1:1-6, preferably 3:1:6.

[0009] Furthermore, the Mg 2+ Hydroxyapatite, doped with Zn 2+ Hydroxyapatite, doped with Sr 2+ Hydroxyapatite, doped Cu 2+ Hydroxyapatite and doped Si 4+ The mass ratio of hydroxyapatite is 83~85:2~4:3~5:1~3:9~11.

[0010] Furthermore, the doped Mg 2+ Mg in hydroxyapatite 2+ The doping mole fraction is 4.5 to 5.5%, preferably 5%.

[0011] Furthermore, the doped Zn 2+ Zn in hydroxyapatite 2+ The doping mole fraction is 4.5 to 5.5%, preferably 5%.

[0012] Furthermore, the doped Sr 2+ Sr in hydroxyapatite 2+ The doping mole fraction is 3 to 4%, preferably 3.2%.

[0013] Furthermore, the doped Cu 2+ Cu in hydroxyapatite 2+ The doping mole fraction is 0.5 to 1.5%, preferably 1%.

[0014] Furthermore, the doped Si 4+ Si in hydroxyapatite 4+The doping mole fraction is 2.5 to 3.5, preferably 3%.

[0015] Furthermore, the mass percentage of citric acid monohydrate in the liquid phase composition is 6-8%, preferably 7%, the mass percentage of disodium hydrogen phosphate dodecahydrate is 7-9%, preferably 8%, and the balance is water.

[0016] Furthermore, the liquid-to-solid ratio of the liquid phase composition to the solid phase composition is 0.35 to 0.45 mL / g.

[0017] The preparation method of tricalcium phosphate bone cement of the present invention comprises the following steps:

[0018] (1) uniformly mixing α-tricalcium phosphate, β-tricalcium phosphate, and five inorganic ion-doped hydroxyapatite to obtain a bone cement solid phase composition;

[0019] (2) preparing a mixed aqueous solution of citric acid monohydrate and disodium hydrogen phosphate dodecahydrate to obtain a bone cement liquid phase composition;

[0020] (3) The solid phase composition and the liquid phase composition are mixed and blended uniformly to obtain the tricalcium phosphate cement doped with hydroxyapatite containing multiple inorganic ions.

[0021] Furthermore, in step (1), the α-tricalcium phosphate, β-tricalcium phosphate and five inorganic ion-doped hydroxyapatite are uniformly mixed by grinding.

[0022] Furthermore, in step (2), the mixed aqueous solution is prepared by stirring and dissolving at room temperature.

[0023] Human bone contains a variety of trace elements, including magnesium, sodium, potassium, strontium, silicon, fluorine, zinc, and copper, among its inorganic components. Therefore, inorganic ions can be introduced into hydroxyapatite based on the trace element content of human bone to produce doped hydroxyapatite, which can then be added to bone cement. This method selects five doped hydroxyapatites based on the trace element content of human bone for inclusion in bone cement. In clinical applications, the inorganic ions are released as the cement degrades, significantly promoting cell proliferation and new bone formation, shortening treatment time.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] (1) The present invention selects a variety of doped hydroxyapatites based on the trace elements contained in human bones and adds them to tricalcium phosphate bone cement, which not only introduces a variety of inorganic ions but also makes the doping content more controllable.

[0026] (2) The tricalcium phosphate bone cement prepared by the present invention with the addition of hydroxyapatite doped with multiple inorganic ions can significantly promote cell proliferation and osteogenic differentiation, and is expected to be applied in the clinical treatment of bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Graph showing the percentage of cells labeled with 5-ethynyl-2'-deoxyuridine (EdU) after co-culturing the extracts of Examples 1 to 6, Comparative Example 1 and Comparative Example 2 with bone marrow mesenchymal stem cells for 3 days;

[0028] Figure 2 Graph showing the expression level of alkaline phosphatase (ALP) gene after 7 days of co-culture of the extracts of Examples 1 to 6, Comparative Example 1 and Comparative Example 2 with bone marrow mesenchymal stem cells. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0030] Example 1:

[0031] (1) Weigh 6g of α-TCP, 3g of β-TCP, and 0.83g of Mg doped with 5% molar fraction. 2+ Hydroxyapatite (denoted as 5Mg-HA), 0.02g doped with 5% Zn 2+ Hydroxyapatite (denoted as 5Zn-HA), 0.03g doped with 3.2% Sr 2+ Hydroxyapatite (denoted as 3.2Sr-HA), 0.01g doped with 1% Cu 2+ Hydroxyapatite (denoted as 1Cu-HA) and 0.11g doped with 3% Si 4+ The hydroxyapatite (denoted as 3Si-HA) is ground and mixed evenly as the solid phase component of bone cement.

[0032] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0033] (3) 0.7 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0034] Example 2:

[0035] The preparation process is the same as in Example 1:

[0036] (1) Weigh 6 g of α-TCP, 2 g of β-TCP, 1.68 g of 5Mg-HA, 0.04 g of 5Zn-HA, 0.06 g of 3.2Sr-HA, 0.02 g of 1Cu-HA, and 0.2 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0037] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0038] (3) 0.8 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0039] Example 3:

[0040] The preparation process is the same as in Example 1:

[0041] (1) Weigh 6 g of α-TCP, 1 g of β-TCP, 2.49 g of 5Mg-HA, 0.08 g of 5Zn-HA, 0.07 g of 3.2Sr-HA, 0.03 g of 1Cu-HA, and 0.33 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0042] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0043] (3) 0.9 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0044] Example 4:

[0045] The preparation process is the same as in Example 1:

[0046] (1) Weigh 5 g of α-TCP, 1 g of β-TCP, 3.32 g of 5Mg-HA, 0.08 g of 5Zn-HA, 0.12 g of 3.2Sr-HA, 0.06 g of 1Cu-HA, and 0.42 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0047] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0048] (3) 0.7 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0049] Example 5:

[0050] The preparation process is the same as in Example 1:

[0051] (1) Weigh 4 g of α-TCP, 1 g of β-TCP, 4.15 g of 5Mg-HA, 0.1 g of 5Zn-HA, 0.13 g of 3.2Sr-HA, 0.07 g of 1Cu-HA, and 0.55 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0052] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0053] (3) 0.8 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0054] Example 6:

[0055] The preparation process is the same as in Example 1:

[0056] (1) Weigh 3 g of α-TCP, 1 g of β-TCP, 4.98 g of 5Mg-HA, 0.12 g of 5Zn-HA, 0.18 g of 3.2Sr-HA, 0.06 g of 1Cu-HA, and 0.66 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0057] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0058] (3) 0.9 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate bone cement doped with hydroxyapatite containing various inorganic ions.

[0059] Comparative Example 1:

[0060] (1) Weigh 3 g of α-TCP, 1 g of β-TCP, and 6 g of undoped hydroxyapatite, grind and mix them evenly to form the solid phase component of bone cement.

[0061] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0062] (3) 0.9 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain tricalcium phosphate / undoped hydroxyapatite bone cement.

[0063] Comparative Example 2:

[0064] (1) Weigh 4 g of α-TCP, 4.98 g of 5Mg-HA, 0.12 g of 5Zn-HA, 0.18 g of 3.2Sr-HA, 0.06 g of 1Cu-HA, and 0.66 g of 3Si-HA, grind and mix them evenly to form the solid phase composition of bone cement.

[0065] (2) Weigh 0.82 g of citric acid monohydrate and 0.94 g of disodium hydrogen phosphate dodecahydrate, add 10 mL of deionized water, and stir to dissolve to prepare a bone cement liquid phase composition.

[0066] (3) 0.9 mL of the liquid phase composition was mixed with 2 g of the solid phase composition and blended evenly to obtain α-tricalcium phosphate / hydroxyapatite bone cement doped with various inorganic ions.

[0067] The bone cements prepared in Examples 1 to 6, Comparative Example 1, and Comparative Example 2 were respectively filled into a mold for curing and curing, and then cured at 37°C and 100% humidity for 48 hours. The extracts of the eight bone cements were co-cultured with bone marrow mesenchymal stem cells (BMSCs) in a 24-well plate for 3 days and stained. The percentage of BMSCs labeled with 5-ethynyl-2'-deoxyuridine (EdU) was counted, as shown in FIG. Figure 1 As shown, Figure 1 The percentage of cells labeled with 5-ethynyl-2'-deoxyuridine (EdU) after co-culturing the extracts of Examples 1 to 6, Comparative Examples 1 and 2 with bone marrow mesenchymal stem cells for 3 days is shown; the expression level of the osteogenic protein alkaline phosphatase (ALP) gene was tested after 7 days of co-culture, as shown in FIG. Figure 2 As shown, Figure 2 The expression level of alkaline phosphatase (ALP) gene after 7 days of co-culture of the extracts of Examples 1 to 6, Comparative Examples 1 and 2 with bone marrow mesenchymal stem cells. Figure 1 and Figure 2 It can be seen that the EdU-labeled cell ratio and ALP gene expression level of the tricalcium phosphate bone cement group prepared in Example 6 with the addition of multiple inorganic ion-doped hydroxyapatite were significantly higher than those of the α-tricalcium phosphate / hydroxyapatite bone cement group prepared in Comparative Example 2, indicating that the effect of adding two tricalcium phosphates is far better than that of adding one tricalcium phosphate. At the same time, the EdU-labeled cell ratio and ALP gene expression level of the tricalcium phosphate bone cement group prepared in Example 6 with the addition of multiple inorganic ion-doped hydroxyapatite were also significantly higher than those of the tricalcium phosphate / undoped hydroxyapatite bone cement group prepared in Comparative Example 1, indicating that the effect of adding multiple inorganic ion-doped hydroxyapatite is far better than that of undoped hydroxyapatite. Therefore, tricalcium phosphate bone cement with the addition of multiple inorganic ion-doped hydroxyapatite can significantly promote cell proliferation and osteogenic differentiation, and is expected to be used in the clinical treatment of bone defects.

Claims

1. A tricalcium phosphate bone cement containing hydroxyapatite doped with multiple inorganic ions, characterized in that: The tricalcium phosphate bone cement is prepared by mixing a solid phase component and a liquid phase component; wherein the solid phase component is α-tricalcium phosphate, β-tricalcium phosphate and hydroxyapatite doped with various inorganic ions, the liquid phase component is a mixed aqueous solution of citric acid monohydrate and disodium hydrogen phosphate dodecahydrate, and the various hydroxyapatite doped with inorganic ions includes Mg doped 2+ Hydroxyapatite, doped with Zn 2+ Hydroxyapatite, doped with Sr 2+ Hydroxyapatite, doped Cu 2+ Hydroxyapatite and doped Si 4+ The mass percentage of α-tricalcium phosphate, β-tricalcium phosphate and hydroxyapatite doped with various inorganic ions is 6-3:3-1:1-6, and the Mg 2+ Hydroxyapatite, doped with Zn 2+ Hydroxyapatite, doped with Sr 2+ Hydroxyapatite, doped Cu 2+ Hydroxyapatite and doped Si 4+ The mass ratio of hydroxyapatite is 83~85:2~4:3~5:1~3:9~11, and the doped Mg 2+ Mg in hydroxyapatite 2+ The doping molar fraction of Zn is 4.5-5.5%, and the doping 2+ Zn in hydroxyapatite 2+ The doping molar fraction of Sr is 4.5-5.5%, and the doped Sr 2+ Sr in hydroxyapatite 2+ The doping molar fraction is 3-4%, and the doped Cu 2+ Cu in hydroxyapatite 2+ The doping mole fraction is 0.5-1.5%, and the doped Si 4+ Si in hydroxyapatite 4+ The doping mole fraction is 2.5~3.5%.

2. The tricalcium phosphate bone cement according to claim 1, wherein The mass percentage of citric acid monohydrate in the liquid phase composition is 6-8%, the mass percentage of disodium hydrogen phosphate dodecahydrate is 7-9%, and the balance is water.

3. The tricalcium phosphate bone cement according to claim 1, wherein The liquid-to-solid ratio of the liquid phase composition to the solid phase composition is 0.35-0.45 mL / g.

4. The method for preparing tricalcium phosphate bone cement according to any one of claims 1 to 3, wherein The following steps are involved: (1) uniformly mixing α-tricalcium phosphate, β-tricalcium phosphate and five inorganic ion-doped hydroxyapatite to obtain a bone cement solid phase composition; (2) preparing a mixed aqueous solution of citric acid monohydrate and disodium hydrogen phosphate dodecahydrate to obtain a bone cement liquid phase composition; (3) The solid phase component and the liquid phase component are mixed and blended uniformly to obtain the tricalcium phosphate cement doped with hydroxyapatite containing multiple inorganic ions.

5. The preparation method according to claim 4, characterized in that In step (1), the α-tricalcium phosphate, β-tricalcium phosphate and five inorganic ion-doped hydroxyapatite are uniformly mixed by grinding.

6. The preparation method according to claim 4, characterized in that In step (2), the mixed aqueous solution is prepared by stirring and dissolving at room temperature.

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