A hydroxyapatite - collagen filling material, its preparation method and application

Abstract

The present invention provides a hydroxyapatite - collagen filling material, a preparation method thereof and an application. The hydroxyapatite - collagen filling material comprises: collagen, p - carboxyphenylphosphonic acid and hydroxyapatite; the p - carboxyphenylphosphonic acid is grafted onto the collagen by forming an amide bond with the collagen; the hydroxyapatite is grafted onto the collagen by the reaction of calcium ions with the p - carboxyphenylphosphonic acid. By grafting the p - carboxyphenylphosphonic acid onto the collagen fibers, the calcium ions further react with the phosphate groups in the p - carboxyphenylphosphonic acid under alkaline conditions to generate hydroxyapatite, avoiding the shedding of calcium; and further improving the toughness of the collagen fibers and the strength of the hydroxyapatite, so that the hydroxyapatite - collagen filling material prepared by the present invention has more excellent mechanical properties.

Description

Technical Field

[0001] The present invention relates to the technical field of bone filling materials, and particularly relates to a hydroxyapatite - collagen filling material, a preparation method thereof, and an application thereof. Background Art

[0002] Natural bone is a composite composed of inorganic minerals and biological macromolecules arranged regularly. Among them, the inorganic substances are mainly hydroxyapatite, and the biological macromolecules mainly include collagen. The two are evenly mixed and combined in an orderly manner. Therefore, the key to the research of artificial bone materials is to obtain artificial bone materials identical to natural bone tissues. This is because if the artificial bone has a similar size to the microcrystalline hydroxyapatite that mineralizes and nucleates on collagen fibers in natural bone tissues, it will help human cells and macromolecules to recognize it, thereby improving the biological activity and biocompatibility of the material.

[0003] There are three common mixing methods: physical mixing method, co - precipitation method, and in - situ deposition method. The physical mixing method usually involves mixing powdered HA and Col colloid solution in a certain proportion as evenly as possible, adding a certain cross - linker to obtain a mixed precipitate, and drying at room temperature or by freeze - drying. Some studies have shown that the HA / Col composite material prepared by the direct blending method has good binding within the scaffold material observed by SEM. The collagen fibers form a network membrane, and HA particles are distributed on it and tightly bound to the Col matrix. However, since this method is a mechanical mixing, it is difficult for the synthesized HA / Col composite material to form a tight bond at the molecular level between the two, and it can only be biomimetic in terms of composition rather than structure.

[0004] The co - precipitation method is to simultaneously drip the Ca(OH)2 suspension obtained by hydrating CaO and the phosphoric acid / collagen solution into a reaction vessel pre - filled with water using an automatic titration device and continuously stir. The temperature during the reaction process is maintained at 40°C, and the pH value is controlled at 8 - 9 by the automatic titration device. After the reaction generates a precipitate, the reaction continues for 24 h, and then it is separated, washed, and dried to obtain HA / Col. The principle of this reaction is that 40°C is the temperature at which collagen releases bound water; when the pH value is 8 - 9, the Zeta potential of collagen is zero, at which time collagen is prone to agglomeration and precipitation, and nano - HA can be stably generated.

[0005] The in - situ deposition method utilizes the principle of self - assembly technology in materials science. First, an HA precursor is generated, and using type I collagen molecules as a template, the mineralization and deposition of calcium phosphate in the liquid phase are induced to generate a bone - like HA / Col composite material. Cui Fuzhai et al. mixed a certain amount of collagen solution and phosphoric acid solution evenly, added the CaCl2 solution drop by drop according to the Ca / P ratio of 5 / 3 and continuously stirred, and then adjusted the pH value to about 7.4 with ammonia water to obtain a precipitate. After separating and washing the precipitate, it was freeze - dried to obtain the HA / Col composite material. Duan Yourong et al. used CaCl2 and Na2 hPO4 is loaded into thermosensitive liposomes of type I collagen. When the temperature reaches its melting transition temperature, calcium phosphate is released and interacts with the type I collagen gel formed by thermally triggered self-assembly to form a mineralized HA / Col composite material.

[0006] In the hydroxyapatite / collagen composite material prepared by the above method, the interfacial binding ability between hydroxyapatite and collagen is relatively low, which easily causes the shedding of hydroxyapatite, thereby changing the properties of the composite material.

[0007] CN114681676A mentions a preparation method of a selenium-doped hydroxyapatite solution for 3D printing, an artificial bone and an artificial periosteum, belonging to the technical field of bone repair. The preparation method includes: obtaining a silk fibroin solution and a collagen solution; mixing the silk fibroin solution and the collagen solution to obtain a double-template solution; adding a phosphorus source solution, a calcium source solution and a selenium source solution to the double-template solution to obtain an added solution; adjusting the pH value of the added solution to 6-8 and then performing solid-liquid separation to obtain a selenium-doped hydroxyapatite solution.

[0008] CN112891621A discloses an artificial bone material loaded with an anti-osteoporosis drug and a method for preparing an artificial bone. The method includes: dissolving hydroxyapatite particles, collagen powder, a dispersant, an anti-osteoporosis drug and trace elements in deionized water, adjusting the pH value, and mixing evenly to obtain a slurry; ball-milling and ultrasonically oscillating the slurry to obtain an ink suspension; loading the ink suspension into the nozzle of a 3D direct writing forming device, connecting a computer to the direct writing forming device, setting the program of the 3D direct writing device and an osteoid structure model, and printing layer by layer in the air by controlling the rheological properties of the ink suspension, and drying to obtain an osteoid structure material loaded with an anti-osteoporosis drug.

[0009] Although the above solutions largely avoid the shedding of calcium ions, they involve the use of multiple methods and the operation is relatively complex. In addition, the collagen fibers only serve as the reaction site for calcium ions and phosphate ions. After the reaction, hydroxyapatite is still deposited on the collagen fibers physically, which will still cause the shedding of calcium ions.

[0010] In view of this, the present invention is specifically proposed. Summary of the Invention

[0011] One of the objectives of the present invention is a hydroxyapatite - collagen filler material. The hydroxyapatite - collagen filler material of the present invention includes: collagen, p - carboxyphenylphosphonic acid, and hydroxyapatite; the p - carboxyphenylphosphonic acid is grafted onto collagen by forming an amide bond with collagen; the hydroxyapatite is grafted onto collagen by the reaction of calcium ions with p - carboxyphenylphosphonic acid. In the present invention, calcium ions react with the phosphate groups in the p - carboxyphenylphosphonic acid grafted onto collagen fibers, avoiding the shedding of calcium ions.

[0012] Another objective of the present invention is to provide a preparation method of the hydroxyapatite - collagen filler material. The preparation method grafts p - carboxyphenylphosphonic acid onto collagen by the amidation reaction of the carboxyl group in p - carboxyphenylphosphonic acid and the amino group in collagen; then, calcium ions and the phosphate groups of p - carboxyphenylphosphonic acid react through an in - situ deposition method to form hydroxyapatite; then, heat treatment is carried out to complete cross - linking, obtaining the hydroxyapatite - collagen filler material. It avoids the shedding of hydroxyapatite from collagen fibers. The p - carboxyphenylphosphonic acid is equivalent to an adhesive, firmly adhering the hydroxyapatite to the collagen fibers.

[0013] In order to achieve the above - mentioned objectives of the present invention, the following technical solutions are specifically adopted:

[0014] In the first aspect, the present invention provides a hydroxyapatite - collagen filler material, which includes: collagen, p - carboxyphenylphosphonic acid, and hydroxyapatite;

[0015] The p - carboxyphenylphosphonic acid is grafted onto collagen by forming an amide bond with collagen;

[0016] The hydroxyapatite is grafted onto collagen by the reaction of calcium ions with p - carboxyphenylphosphonic acid.

[0017] In the present invention, the structure of the hydroxyapatite - collagen filler material avoids the shedding of hydroxyapatite from collagen fibers. The p - carboxyphenylphosphonic acid is equivalent to an adhesive, firmly adhering the hydroxyapatite to the collagen fibers; and the above - mentioned structure further improves the toughness of collagen fibers and the strength of hydroxyapatite, making the mechanical properties of the hydroxyapatite - collagen filler material prepared by the present invention more excellent.

[0018] Preferably, the mass ratio of collagen, p - carboxyphenylphosphonic acid, and hydroxyapatite is 100:(0.3 - 0.4):(30 - 50);

[0019] Among them, "0.3 - 0.4" can be, for example, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, etc.;

[0020] Among them, "30 - 50" can be, for example, 30, 35, 40, 45, 50, etc.

[0021] Preferably, the raw materials for preparing the hydroxyapatite - collagen filler include the following components: collagen slurry, p - carboxyphenylphosphonic acid, and calcium salt.

[0022] Preferably, the molecular weight of the collagen is 10 - 200 kDa, and can be, for example, 10 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, etc., and is preferably 120 - 140 kDa.

[0023] Preferably, the concentration of the collagen slurry is 2 - 8 g / L, and can be, for example, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, etc.

[0024] Preferably, the calcium salt is calcium chloride.

[0025] Preferably, the raw materials for preparing the hydroxyapatite - collagen filler further include a halogenating reagent.

[0026] Preferably, the halogenating reagent is a halogenated phosphorus, and preferably phosphorus pentachloride.

[0027] Preferably, the mass ratio of the collagen slurry, halogenating reagent, p - carboxyphenylphosphonic acid, and calcium salt is 100:(3 - 5):(0.3 - 0.4):(0.3 - 0.5);

[0028] Among them, "3 - 5" can be, for example, 3, 3.5, 4, 4.5, 5, etc.;

[0029] Among them, "0.3 - 0.4" can be, for example, 0.3, 0.32, 0.34, 0.36, 0.38, 0.4, etc.;

[0030] Among them, "0.3 - 0.5" can be, for example, 0.3, 0.35, 0.4, 0.45, 0.5, etc.

[0031] Preferably, the raw materials for preparing the hydroxyapatite - collagen filler further include a basic pH regulator.

[0032] Preferably, the basic pH regulator is an aqueous sodium hydroxide solution of 1 - 20 wt% (which can be, for example, 1 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt%, etc.).

[0033] In a second aspect, the present invention provides a method for preparing the hydroxyapatite - collagen filler as described in the first aspect, and the preparation method includes the following steps:

[0034] The carboxyphenylphosphonic acid is grafted onto collagen by an amidation reaction between the carboxyl group in the carboxyphenylphosphonic acid and the amino group in the collagen; then, calcium ions react with the phosphate groups of the carboxyphenylphosphonic acid through an in-situ deposition method to form hydroxyapatite; and then heat treatment is carried out to complete crosslinking, obtaining a hydroxyapatite-collagen filler material.

[0035] In the present invention, first, an amidation reaction is utilized to graft the carboxyphenylphosphonic acid onto the collagen fibers by an amidation reaction between the carboxyl group in the carboxyphenylphosphonic acid and the amino group in the collagen; then, calcium ions react with the phosphate groups on the carboxyphenylphosphonic acid through an in-situ deposition method; and finally, heat treatment after drying is carried out to complete crosslinking. The carboxyphenylphosphonic acid acts as an adhesive, not only preventing the hydroxyapatite from falling off the collagen fibers, but also firmly adhering the hydroxyapatite to the collagen fibers; and since the carboxyl group and the phosphate group are in the para position, the composite material obtained after grafting and then crosslinking further improves the toughness of the collagen fibers and the strength of the hydroxyapatite, making the mechanical properties of the hydroxyapatite-collagen material prepared by the present invention more excellent.

[0036] Preferably, the preparation method of the hydroxyapatite-collagen filler material specifically includes the following steps:

[0037] (1) Mix the collagen slurry and the halogenating reagent, and carry out a halogenation reaction under alkaline conditions;

[0038] (2) Mix the reaction solution obtained in step (1) with the carboxyphenylphosphonic acid to carry out an amidation reaction;

[0039] (3) Mix the reaction solution obtained in step (2) with the calcium chloride solution to react to form hydroxyapatite;

[0040] (4) Dry the reaction solution obtained in step (3) and then carry out heat treatment to complete crosslinking, obtaining a hydroxyapatite-collagen filler material.

[0041] Preferably, in step (1), the mixing method is: dropping the halogenating reagent into the collagen slurry.

[0042] Preferably, in step (1), the temperature of the halogenation reaction is 10 - 30 °C, such as 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, etc., and the time of the halogenation reaction is 1 - 3 h, such as 1 h, 1.5 h, 1.8 h, 2 h, 2.2 h, 2.5 h, 3 h, etc.

[0043] Preferably, in step (1), the halogenation reaction needs to be carried out in a reaction system with a pH of 8 - 9.

[0044] Preferably, in step (2), the mixing method is: adding 4-carboxyphenylphosphonic acid to the reaction solution obtained in step (1).

[0045] Preferably, in step (2), the temperature of the amidation reaction is 40 - 60 °C, such as 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, etc., and the time of the amidation reaction is 6 - 10 h, such as 6 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 10 h, etc.

[0046] Preferably, in step (3), the pH of the reaction solution needs to be adjusted to 11 - 12 before mixing, such as 11, 11.2, 11.4, 11.6, 11.8, 12, etc.

[0047] Preferably, in step (3), the mixing method is: dropping a calcium chloride solution into the reaction solution obtained in step (2).

[0048] Preferably, the dropping flow rate is 2 - 2.5 g / min, such as 2 g / min, 2.1 g / min, 2.2 g / min, 2.3 g / min, 2.4 g / min, 2.5 g / min, etc.

[0049] Preferably, the concentration of the calcium chloride solution is 0.06 - 0.12 mol / L, such as 0.06 mol / L, 0.08 mol / L, 0.10 mol / L, 0.12 mol / L, etc.

[0050] Preferably, in step (3), the temperature of the reaction is 10 - 40 °C, such as 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, etc., and the reaction time is 0.2 - 0.6 h, such as 0.2 h, 0.3 h, 0.4 h, 0.5 h, 0.6 h, etc.

[0051] Preferably, in step (4), freeze-drying is used for drying.

[0052] Preferably, the lyophilization includes a first freezing stage, a second freezing stage, and a room temperature stage that are carried out in sequence. Among them, the temperature of the first freezing stage is -40 to -10 °C (for example, it can be -40 °C, -35 °C, -30 °C, -25 °C, -20 °C, -15 °C, -10 °C, etc.), the holding time is 1-6 h (for example, it can be 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, etc.), the temperature of the second freezing stage is -10 to 0 °C (for example, it can be -10 °C, -8 °C, -6 °C, -4 °C, -2 °C, 0 °C, etc.), the holding time is 12-48 h (for example, it can be 12 h, 16 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, etc.), and the holding time of the room temperature stage is 1-2 h (for example, it can be 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.).

[0053] Preferably, the temperature of the first freezing stage is -30 to -20 °C, for example, it can be -30 °C, -28 °C, -26 °C, -24 °C, -22 °C, -20 °C, etc.

[0054] Preferably, the holding time of the second freezing stage is 24-36 h, for example, it can be 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, etc.

[0055] Preferably, in step (4), the temperature of the heat treatment is 110-130 °C, for example, it can be 110 °C, 115 °C, 120 °C, 125 °C, 130 °C, etc., and the time of the heat treatment is 20-40 h, for example, it can be 20 h, 25 h, 30 h, 35 h, 40 h, etc.

[0056] As a preferred technical solution of the present invention, the preparation method of the hydroxyapatite-collagen filling material includes the following steps:

[0057] (1) Add a halogenating reagent dropwise to the collagen slurry, react at 10-30 °C for 1-3 h, and use an alkaline pH regulator to control the pH of the reaction system between 8-9 during the reaction;

[0058] (2) Add p-carboxyphenylphosphoric acid to the reaction solution obtained in step (1), and react at 40-60 °C for 6-10 h;

[0059] (3) Use an alkaline pH regulator to adjust the pH of the reaction solution obtained in step (2) to 11-12, and add a calcium chloride solution thereto at a flow rate of 2-2.5 g / min under stirring, and react at 10-40 °C for 0.2-0.6 h;

[0060] (4) The reaction solution obtained in step (3) is freeze-dried at -50 to -10 °C for 36 - 56 h, and then heat-treated at 110 - 130 °C for 20 - 40 h to obtain the hydroxyapatite-collagen filler material.

[0061] In a third aspect, the present invention provides an application of the hydroxyapatite-collagen filler material described in the first aspect in the preparation of a bone filler material.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] The structure of the hydroxyapatite-collagen filler material provided by the present invention prevents hydroxyapatite from falling off the collagen fibers. Carboxyphenylphosphoric acid acts as an adhesive, firmly bonding the hydroxyapatite to the collagen fibers. Moreover, the above structure further improves the toughness of the collagen fibers and the strength of the hydroxyapatite, making the mechanical properties of the hydroxyapatite-collagen filler material prepared by the present invention more excellent. Detailed implementation manners

[0064] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0065] Generally, the nomenclature and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, are those well known and commonly used in the art.

[0066] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0067] The present invention will be further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0068] Example 1

[0069] This example provides a hydroxyapatite - collagen filling material, and the hydroxyapatite - collagen filling material is prepared by the following method:

[0070] (1) Take 100 g of collagen slurry (collagen molecular weight is 130 kD, concentration is 4 g / L), place it on a magnetic stirrer, and while stirring, slowly drop 3.6 mL of PCl5, react at 20 °C for 2 h, and use 10% (mass fraction) NaOH solution to control the pH of the reaction system at 8.5 during the reaction;

[0071] (2) Add 0.33 g of p - carboxyphenylphosphoric acid to the above reaction solution, and react at 50 °C for 8 h,

[0072] (3) Adjust the pH of the above reaction solution to 11.4 with sodium hydroxide, and while stirring, add a certain amount (30 mL, 0.1 mol / L) of calcium chloride solution thereto at a flow rate of 2.3 g / min, and react at 30 °C for 0.3 h;

[0073] (4) Lyophilize the above reaction solution at - 40 °C for 40 h, and then heat - treat it at 120 °C for 30 h to obtain the hydroxyapatite - collagen filling material.

[0074] Comparative Example 1

[0075] This comparative example provides a hydroxyapatite - collagen filling material, and the hydroxyapatite - collagen filling material is prepared by the following method:

[0076] Take 100 g of collagen slurry (collagen molecular weight is 130 kD, concentration is 4 g / L), adjust the pH of the solution to 7 with 1 mol / L sodium hydroxide, and while stirring, simultaneously drop 30 mL of calcium chloride and sodium phosphate solutions with concentrations of 0.1 mol / L and 0.06 mol / L respectively at flow rates of 1.8 g / min and 1.6 g / min, stir for 3 h, after the reaction ends, lyophilize at - 40 °C for 40 h, and then heat - treat it at 120 °C for 30 h to obtain the hydroxyapatite - collagen filling material.

[0077] Comparative Example 2

[0078] This comparative example provides a hydroxyapatite - collagen filling material, and the hydroxyapatite - collagen filling material is prepared by the following method:

[0079] (1) Take 100 g of collagen slurry (collagen molecular weight is 120 kD, concentration is 4 g / L), place it on a magnetic stirrer, and slowly add 3.6 mL of PCl5 dropwise while stirring. React at 25 °C for 2 h, and use a 12% NaOH solution by mass fraction to control the pH of the reaction system at 8 during the reaction;

[0080] (2) Add 0.33 g of m-carboxyphenylphosphonic acid to the above reaction solution, and react at 48 °C for 8.5 h,

[0081] (3) Adjust the pH of the above reaction solution to 11.2 with sodium hydroxide, and add a certain amount (30 mL, 0.1 mol / L) of calcium chloride solution to it at a flow rate of 2.4 g / min while stirring. React at 30 °C for 0.3 h;

[0082] (4) Lyophilize the above reaction solution at -40 °C for 40 h, and then heat-treat it at 110 °C for 32 h to obtain the hydroxyapatite-collagen filling material.

[0083] Comparative Example 3

[0084] This comparative example provides a hydroxyapatite-collagen filling material, and the hydroxyapatite-collagen filling material is prepared by the following method:

[0085] (1) Take 100 g of collagen slurry (collagen molecular weight is 120 kD, concentration is 4 g / L), place it on a magnetic stirrer, and slowly add 3.6 mL of PCl5 dropwise while stirring. React at 25 °C for 2 h, and use a 12% NaOH solution by mass fraction to control the pH of the reaction system at 8 during the reaction;

[0086] (2) Add 0.33 g of 6-carboxyhexylphosphocholine to the above reaction solution, and react at 48 °C for 8.5 h,

[0087] (3) Adjust the pH of the above reaction solution to 11.2 with sodium hydroxide, and add a certain amount (30 mL, 0.1 mol / L) of calcium chloride solution to it at a flow rate of 2.4 g / min while stirring. React at 30 °C for 0.3 h;

[0088] (4) Lyophilize the above reaction solution at -40 °C for 40 h, and then heat-treat it at 110 °C for 32 h to obtain the hydroxyapatite-collagen filling material.

[0089] Test Example 1

[0090] Calcium ion loading test

[0091] Test samples: Hydroxyapatite - collagen filling materials prepared in Example 1, and hydroxyapatite - collagen filling materials prepared in Comparative Examples 1 - 3;

[0092] Test methods: Record the content of Ca in the calcium salts added in Example 1 and Comparative Examples 1 - 3 respectively; 2+ Then, use an inductively coupled plasma optical emission spectrometer (ICP - OES) to test the content of Ca in the filling materials prepared in Example 1 and Comparative Examples 1 - 3; 2+ Content;

[0093] The specific test results are shown in Table 1:

[0094] Table 1

[0095] Group <![CDATA[Ca 2+ feed rate]]> <![CDATA[Ca 2+ content]]> Example 1 45％ 48％ Comparative Example 1 45％ 32％ Comparative Example 2 45％ 21％ Comparative Example 3 45％ 22％

[0096] It can be seen from the test results in Table 1 that the content of Ca in the test leaching solution of the present invention is 48%, indicating that by grafting p - carboxyphenylphosphonic acid onto collagen fibers, calcium ions further react with the phosphate groups in p - carboxyphenylphosphonic acid under alkaline conditions to form hydroxyapatite, avoiding the shedding of calcium. 2+ Content;

[0097] Test Example 2

[0098] Mechanical property test

[0099] Test samples: Hydroxyapatite - collagen filling materials prepared in Example 1, and hydroxyapatite - collagen filling materials prepared in Comparative Examples 1 - 3;

[0100] Test methods: Make the test specimens into cylindrical shapes with a diameter of 10 mm and a height of 20 mm, measure their compressive resistance on a force measuring instrument, and calculate the compressive strength.

[0101] The specific test results are shown in Table 3:

[0102] Table 2

[0103]

[0104]

[0105] It can be seen from the test data in Table 3 that the compressive strength of the hydroxyapatite - collagen filling material prepared in the present invention is 5.9 - 6.5 MPa, indicating that the collagen - hydroxyapatite artificial bone prepared in the present invention has good mechanical properties. Since p - carboxyphenylphosphonic acid is grafted onto collagen by forming an amide bond with collagen, the structure further improves the toughness of collagen fibers and the strength of hydroxyapatite, making the mechanical properties of the hydroxyapatite - collagen filling material prepared in the present invention more excellent.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A hydroxyapatite - collagen filling material, characterized in that, The hydroxyapatite - collagen filler material includes: collagen, p - carboxyphenylphosphonic acid, and hydroxyapatite; The p - carboxyphenylphosphonic acid is grafted onto collagen by forming an amide bond with collagen; The hydroxyapatite is grafted onto collagen through the reaction of calcium ions and p - carboxyphenylphosphonic acid.

2. The hydroxyapatite - collagen filling material according to claim 1, characterized in that, The mass ratio of the collagen, p - carboxyphenylphosphonic acid, and hydroxyapatite is 100:(0.3 - 0.4):(30 - 50).

3. The hydroxyapatite - collagen filling material according to claim 1, characterized in that, The raw materials for preparing the hydroxyapatite - collagen filler material include the following components: collagen slurry, p - carboxyphenylphosphonic acid, and calcium salt.

4. The hydroxyapatite - collagen filling material according to claim 3, wherein The molecular weight of the collagen in the collagen slurry is 10 - 200 kDa.

5. The hydroxyapatite-collagen filling material according to claim 4, wherein The molecular weight of the collagen in the collagen slurry is 120 - 140 kDa.

6. The hydroxyapatite - collagen filling material according to claim 3, characterized in that, The concentration of the collagen slurry is 2 - 8 g / L.

7. The hydroxyapatite - collagen filler according to claim 3, characterized in that, The calcium salt is calcium chloride.

8. The hydroxyapatite - collagen filling material according to claim 3, wherein, The raw materials for preparing the hydroxyapatite - collagen filler material further include a halogenating reagent.

9. The hydroxyapatite - collagen filling material according to claim 8, wherein, The halogenating reagent is a phosphorus halide.

10. The hydroxyapatite - collagen filling material according to claim 9, characterized in that, The halogenating reagent is phosphorus pentachloride.

11. The hydroxyapatite - collagen filling material according to claim 8, characterized in that, The mass ratio of the collagen slurry, halogenating reagent, p - carboxyphenylphosphonic acid, and calcium salt is 100:(3 - 5):(0.3 - 0.4):(0.3 - 0.5).

12. The hydroxyapatite - collagen filling material according to claim 3, characterized in that, The raw materials for preparing the hydroxyapatite - collagen filler material further include a basic pH regulator.

13. The hydroxyapatite - collagen filling material according to claim 12, wherein The basic pH regulator is a 1 - 20 wt% aqueous sodium hydroxide solution.

14. A method for preparing the hydroxyapatite - collagen filler according to any one of claims 1 - 13, characterized in that, The preparation method includes the following steps: By the amidation reaction of the carboxyl group in p - carboxyphenylphosphonic acid and the amino group in collagen, graft p - carboxyphenylphosphonic acid onto collagen; then through the in - situ deposition method, make calcium ions react with the phosphate group of p - carboxyphenylphosphonic acid to form hydroxyapatite; then perform heat treatment to complete cross - linking, and obtain the hydroxyapatite - collagen filler material.

15. The preparation method of the hydroxyapatite - collagen filling material according to claim 14, wherein, The specific preparation method includes the following steps: (1) Mix the collagen slurry and the halogenating reagent, and under alkaline conditions, carry out a halogenation reaction; (2) Mix the reaction solution obtained in step (1) with p - carboxyphenylphosphonic acid to carry out an amidation reaction; (3) Mix the reaction solution obtained in step (2) with a calcium chloride solution, and react to form hydroxyapatite; (4) Dry the reaction solution obtained in step (3) and then perform heat treatment to complete cross - linking, and obtain the hydroxyapatite - collagen filler material.

16. The preparation method of the hydroxyapatite-collagen filling material according to claim 15, characterized in that, In step (1), the mixing method is: dropwise add the halogenating reagent to the collagen slurry.

17. The preparation method of the hydroxyapatite-collagen filling material according to claim 15, wherein, In step (1), the temperature of the halogenation reaction is 10 - 30 °C, and the time of the halogenation reaction is 1 - 3 h.

18. The preparation method of the hydroxyapatite-collagen filler according to claim 15, wherein, In step (1), the halogenation reaction needs to be carried out in a reaction system with a pH of 8 - 9.

19. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, wherein, In step (2), the mixing method is: add p - carboxyphenylphosphonic acid to the reaction solution obtained in step (1).

20. The preparation method of the hydroxyapatite - collagen filler according to claim 15, characterized in that, In step (2), the temperature of the amidation reaction is 40 - 60 °C, and the time of the amidation reaction is 6 - 10 h.

21. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, wherein, In step (3), before mixing, the pH of the reaction solution needs to be adjusted to 11 - 12.

22. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, wherein, In step (3), the mixing method is: dropwise add the calcium chloride solution to the reaction solution obtained in step (2).

23. The preparation method of the hydroxyapatite - collagen filling material according to claim 22, wherein, In step (3), the dropping flow rate is 2 - 2.5 g / min.

24. The preparation method of the hydroxyapatite - collagen filler according to claim 22, characterized in that, The concentration of the calcium chloride solution is 0.06 - 0.12 mol / L.

25. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, characterized in that, In step (3), the temperature of the reaction is 10 - 40 °C, and the reaction time is 0.2 - 0.6 h.

26. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, characterized in that, In step (4), the drying is carried out by freeze-drying.

27. The preparation method of the hydroxyapatite - collagen filling material according to claim 26, characterized in that, In step (4), the freeze-drying includes a first freezing stage, a second freezing stage, and a room temperature stage that are carried out in sequence. Among them, the temperature of the first freezing stage is -40~-10 °C, and the holding time is 1 - 6 h. The temperature of the second freezing stage is -10~0 °C, and the holding time is 12 - 48 h. The holding time of the room temperature stage is 1 - 2 h.

28. The preparation method of the hydroxyapatite - collagen filling material according to claim 27, characterized in that, The temperature of the first freezing stage is -30~-20 °C.

29. The preparation method of the hydroxyapatite - collagen filling material according to claim 27, wherein The holding time of the second freezing stage is 24 - 36 h.

30. The preparation method of the hydroxyapatite - collagen filling material according to claim 15, wherein, In step (4), the temperature of the heat treatment is 110 - 130 °C, and the time of the heat treatment is 20 - 40 h.

31. Use of a hydroxyapatite - collagen filling material according to any one of claims 1 - 13 in the preparation of a bone filling material.

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