Artificial bone composite material and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to an artificial bone composite material and a preparation method thereof. The preparation method of the artificial bone composite material comprises the following steps: sequentially adding a CaCl2 solution and a NaH2PO4 solution into a collagen solution in proportion. And adjusting the pH value to 7.4, and collecting white jelly. Dispersing the white jelly in distilled water to obtain a colloid I; respectively adding the colloid I and the modified polyether-ether-ketone powder into a dichloromethane solution containing poly-L-lactic acid in proportion to obtain a mixed solution II, and then adding an ethanol solution for precipitation; and removing the precipitate in the mixed solution II to obtain the slurry. Injecting the slurry into a mold to obtain an intermediate I, and soaking the intermediate I in an EDC / NHS ethanol solution for crosslinking; according to the preparation method, the artificial bone composite material with high mechanical strength and good biocompatibility can be prepared, the defect that the biocompatibility and mechanical strength resistance of the artificial bone composite material cannot be obtained at the same time in the prior art can be overcome, and the practicability of the artificial bone composite material is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to an artificial bone composite material and a preparation method thereof. Background Art

[0002] Bone defects are usually caused by trauma, osteomyelitis, bone tumor resection, and congenital bone defects. Severe bone defects will affect the normal life of patients. With the continuous increase in bone defect diseases, the preparation of bone defect repair materials has become one of the current research hotspots. According to statistics, about 20 million bone defect patients need to receive repair treatment globally every year. At present, autologous bone transplantation and allogeneic bone transplantation are usually regarded as the preferred methods for treating bone defects in clinical practice. However, this method has the following defects: For autologous bone transplantation, autologous bone is the clinical "gold standard" for bone defect treatment, but its application is limited due to the limited source of autologous bone and the secondary injury it causes to patients. For allogeneic bone transplantation, allogeneic bone transplantation may induce immune rejection reactions in the body, and problems such as slow healing of the transplanted bone are likely to occur after transplantation. Based on this, people use artificial synthetic biomaterials to replace traditional bone transplantation methods. Implanting artificial bone made of artificial synthetic biomaterials into the human body can not only not damage the health of the human body itself, but also solve the problem of rejection reactions that can occur in allogeneic bone transplantation in the prior art.

[0003] Among them, biocompatibility and anti-mechanical strength are two important indicators for measuring whether a bone repair material is excellent. For artificial bone synthesized by existing artificial synthetic biomaterials, its biocompatibility and anti-mechanical strength cannot be achieved at the same time, which results in the poor practicability of the prepared artificial bone. Summary of the Invention

[0004] In order to solve the technical problem that the artificial bone synthesized by artificial synthetic biomaterials in the prior art cannot achieve both biocompatibility and anti-mechanical strength, resulting in poor practicability of the prepared artificial bone, the present invention provides an artificial bone composite material and a preparation method thereof.

[0005] The present invention is achieved by the following technical solutions: A preparation method of an artificial bone composite material includes the following steps:

[0006] Add a CaCl2 solution and a NaH2PO4 solution to the collagen solution in sequence according to a ratio to obtain a first mixed solution; the molar ratio of calcium ions to phosphate ions in the first mixed solution is 1.67.

[0007] Then add an alkali solution to the first mixed solution to adjust the pH of the first mixed solution to 7.4, mix evenly, and centrifuge to collect the white jelly-like substance.

[0008] The white gel is fully dispersed in distilled water and ultrasonicated to obtain Colloid 1. Then, Colloid 1 and the modified polyetheretherketone powder are respectively added to a dichloromethane solution containing poly-L-lactic acid in proportion, and after mixing evenly by high-speed shearing, a mixed solution 2 is obtained.

[0009] An ethanol solution is added to the mixed solution 2 until the precipitate in the mixed solution 2 is completely precipitated. The precipitate in the mixed solution 2 is removed to obtain a slurry. The slurry is injected into a mold for vacuum degassing and freeze-dried at -20°C to -60°C to obtain Intermediate 1.

[0010] Intermediate 1 is soaked in a 95% ethanol solution containing 1 mol / L EDC and 0.25 mol / L NHS for crosslinking, washed with deionized water, and then freeze-dried to obtain the artificial bone composite material.

[0011] As a further improvement of the present invention, the modified polyetheretherketone is prepared by the following steps: sulfonated polyetheretherketone is prepared by chemical etching with sulfuric acid, and then the sulfonated polyetheretherketone is soaked in a dopamine solution for a light-avoiding reaction to obtain the modified polyetheretherketone.

[0012] As a further improvement of the present invention, the modified polyetheretherketone powder is obtained by pulverizing the modified polyetheretherketone to particles with a particle size of 0.5 nm to 1 nm by an ultramicro pulverizer.

[0013] As a further improvement of the present invention, the preparation method of sulfonated polyetheretherketone is as follows:

[0014] (1) Prepare a polyetheretherketone wafer and polish it successively with 600#, 800#, 1000#, 1200# and 1500# silicon sandpapers.

[0015] (2) The polished polyetheretherketone wafer is ultrasonically rinsed successively with propanol, absolute ethanol and deionized water, and then dried at 50°C for standby.

[0016] (3) Then, the dried polyetheretherketone is soaked in concentrated sulfuric acid at room temperature, and after 6 minutes, the acidified polyetheretherketone is taken out. Then, the acidified polyetheretherketone is ultrasonically oscillated successively in acetone, absolute ethanol and deionized water, and then dried in air to obtain sulfonated polyetheretherketone.

[0017] As a further improvement of the present invention, the dopamine solution is a 10 mM Tris-HCl buffer solution with a pH of 8.5 and a dopamine content of 2 g / L.

[0018] As a further improvement of the present invention, the concentration of the CaCl2 solution is 0.2 mol / L; the concentration of the collagen solution is 1.1 mg / mL, and the volume ratio of the collagen solution to the CaCl2 solution is 1:15 to 1:20. The concentration of NaH2PO4 is 1 mol to 1.5 mol / L, and the volume ratio of NaH2PO4 to the CaCl2 solution is 1:15 to 1:20.

[0019] As a further improvement of the present invention, the mold is a cylindrical polytetrafluoroethylene mold with a diameter of 1.2 cm and a height of 2 cm. The height of the slurry added to the mold is 1 cm.

[0020] As a further improvement of the present invention, the collagen is type I bovine collagen. The mass concentration of poly(L-lactic acid) in the dichloromethane solution containing poly(L-lactic acid) is 4% to 5%, and the volume ratio of the dichloromethane solution to colloid I is 1:3 to 1:4.

[0021] As a further improvement of the present invention, the alkali solution is any one of NaOH or Ca(OH)2.

[0022] As a further improvement of the present invention, the collagen solution is prepared by dissolving type I bovine collagen in an acetic acid solution with a concentration of 0.2 mol / L; and the solid-liquid ratio of type I bovine collagen to the acetic acid solution is 10 mg:1 mL.

[0023] The present invention also includes an artificial bone composite material, which is prepared by using the preparation method of the above artificial bone composite material. The compressive strength of the artificial bone composite material is greater than or equal to 13.4 MPa. The water contact angle on the surface of the artificial bone composite material is 25.6° ± 1.23°.

[0024] The technical solution provided by the present invention has the following beneficial effects:

[0025] (1) The artificial bone composite material provided by the present invention, by using modified polyetheretherketone, collagen, hydroxyapatite, and poly(L-lactic acid) as the main raw materials, can prepare an artificial bone composite material with high mechanical strength and good biocompatibility. It can solve the defect that the biocompatibility and anti-mechanical strength of the artificial bone composite material in the prior art cannot be both achieved, thereby improving the practicality of the prepared artificial bone composite material.

[0026] (2) The artificial bone composite material provided by the present invention, by using modified polyetheretherketone, collagen, hydroxyapatite, and poly(L-lactic acid) as the main raw materials, can synthesize a biomaterial with good biocompatibility, biodegradability, and osteogenic activity. And this biomaterial can significantly promote the proliferation and adhesion of cells, thereby promoting the reproduction of bone cells in vivo.

[0027] (3) The artificial bone composite material provided by the present invention is crosslinked by EDC / NHS during the preparation process, so that the prepared artificial bone composite material can maintain good biocompatibility, leave no toxic residues, reduce the risk of adverse biological reactions, and is particularly suitable for biomedical applications. In addition, this crosslinking process also improves the mechanical strength and stability of the collagen matrix, and enhances the anti-mechanical stress and enzyme degradation capabilities of the prepared artificial bone composite material. Description of the Drawings

[0028] Figure 1 It is a flowchart for the preparation of the artificial bone composite material provided in Example 2 of the present invention.

[0029] Figure 2 It is an external structure diagram of the artificial bone composite material provided in Example 2 of the present invention.

[0030] Figure 3 It is an external structure diagram of the artificial bone composite material provided in Comparative Example 2 of the present invention.

[0031] Figure 4 It is an infrared spectrum diagram of the artificial bone composite material provided in Example 2 of the present invention.

[0032] Figure 5 It is a schematic diagram when the artificial bone composite material provided in Example 2 of the present invention is placed on a compressive strength testing machine.

[0033] Figure 6 It is a relationship curve graph between the displacement of the artificial bone composite material provided in Example 2 of the present invention and the pressure applied by the compressive strength testing machine.

[0034] Figure 7 It is an X-ray diffraction pattern of the crystal type of the artificial bone composite material provided in Example 2 of the present invention.

[0035] Figure 8 It is a thermogravimetric analysis curve graph of the artificial bone composite material provided in Example 2 of the present invention.

[0036] Figure 9 It is an XPS spectrum of polyetheretherketone, sulfonated polyetheretherketone, and modified polyetheretherketone in Example 1 of the present invention.

[0037] Figure 10 It is an electron microscope image of polyetheretherketone, sulfonated polyetheretherketone, and modified polyetheretherketone in Example 1 of the present invention after co-culturing with cells for 12 h respectively.

[0038] Figure 11 It is a bar graph of the cell proliferation of polyetheretherketone, sulfonated polyetheretherketone, and modified polyetheretherketone in Example 1 of the present invention after co-culturing with cells for 1 day, 2 days, and 3 days respectively.

[0039] Figure 12 SEM scanning electron micrograph of the artificial bone composite material provided in Comparative Example 2 of the present invention. Detailed implementation manners

[0040] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0041] Embodiment 1

[0042] This embodiment provides a preparation method of an artificial bone composite material, which includes the following steps:

[0043] (1) Preparation of a collagen-hydroxyapatite composite

[0044] (1.1) Dissolve 1.1 g of collagen in 0.2 mol / L acetic acid solution to prepare a 1.1 mg / mL collagen solution.

[0045] (1.2) Add 50 mL of 0.2 mol / L CaCl2 solution to 1000 mL of 1.1 mg / mL collagen solution under stirring conditions.

[0046] (1.3) Then slowly add 50 mL of 1.2 mol / L NaH2PO4 solution to the collagen solution in step (1.2) to obtain a first mixed solution, and the molar ratio of calcium ions to phosphate ions in the first mixed solution is 1.67.

[0047] (1.4) Then add NaOH solution to the first mixed solution to adjust the pH value of the first mixed solution to 7.4. Then stir for 72 h, and collect the white jelly in the first mixed solution by centrifugation. Then wash the collected white jelly with ultrapure water three times to remove the soluble salts in the white jelly.

[0048] The white jelly obtained by the above method is the collagen-hydroxyapatite composite.

[0049] In this step, collagen (Col) is a natural protein found in the extracellular matrix of bone cells. It has high biocompatibility, biological activity, hydrophilicity, low antigenicity and degradability, and can promote the migration and attachment of bone cells. Type I collagen has the function of regulating and promoting the growth of bone cells.

[0050] In this embodiment, type I bovine collagen can be selected. Type I bovine collagen has a wider source and higher biocompatibility, making the artificial bone composite material prepared from type I bovine collagen have a low cost and no adverse reactions to the human body. It can be understood that in this embodiment, by using type I bovine collagen as a raw material to prepare the artificial bone composite material, after being implanted into the human body, type I bovine collagen can be degraded into amino acids, thereby providing sufficient nutrition for bone tissue cells, stimulating osteoblasts, fibroblasts, etc. to spread and proliferate on its surface, and then promoting bone formation.

[0051] And hydroxyapatite (Ca 10 (PO4)6(OH)2) is the main mineral component of vertebrate bones and teeth, and has a structure and biochemical properties similar to the mineral composition of human bone tissue. It is the most important inorganic substance in human bone tissue, with a length of 200 nm to 400 nm and a thickness of 15 nm to 30 nm. By selecting hydroxyapatite as the raw material for the artificial bone, after the artificial bone is implanted into the human body, hydroxyapatite can closely combine with the surrounding tissues in a short time, thereby improving the biocompatibility of the prepared artificial bone composite material with human tissues after implantation.

[0052] (II) Preparation of poly(lactic acid)-modified poly(ether ether ketone)-collagen-hydroxyapatite composite

[0053] The white jelly-like substance (i.e., the collagen-hydroxyapatite composite) prepared in step (I) is fully dispersed in distilled water. After ultrasonic treatment for 30 min, colloid I is obtained. Then, colloid I and the modified poly(ether ether ketone) powder are respectively added to a dichloromethane solution containing L-lactic acid with a volume of 176 mL. The solid-liquid ratio of the modified poly(ether ether ketone) powder to the ultrasonically treated colloid I is 1 mg:1 mL.

[0054] Then, it is stirred by a high-speed shearer at 2500 r / min to obtain mixture II. At the same time, a large amount of ethanol is added to mixture II until the precipitate in mixture II precipitates completely. Then, the clear liquid in mixture II is removed to remove the dichloromethane in mixture II, and a precipitate is obtained. Then, the obtained precipitate is washed and separated with deionized water to form a uniform and viscous slurry. The slurry is injected into a polytetrafluoroethylene cylindrical mold with a diameter of 1.2 cm and a height of 2 cm, vacuum degassed, and pre-cooled overnight at -20 °C to -60 °C for 12 h, and then freeze-dried at -20 °C to -60 °C for 36 h. Then, the freeze-dried sample is soaked in a 95% ethanol solution containing 1 mol / L EDC and 0.25 mol / L NHS for crosslinking, washed with deionized water, and then freeze-dried to obtain the artificial bone composite material.

[0055] Among them, in this step, the modified polyetheretherketone powder can be pulverized into particles with a particle size of 0.5 nm to 1 nm by an ultrafine pulverizer.

[0056] It can be understood that in this embodiment, the modified polyetheretherketone can be prepared through the following steps: First, sulfonated polyetheretherketone can be prepared by chemical etching with sulfuric acid, and then the prepared sulfonated polyetheretherketone is immersed in a dopamine solution for a light-shielded reaction, thus obtaining the modified polyetheretherketone.

[0057] The preparation method of sulfonated polyetheretherketone can be referred to as follows: (1) Prepare a polyetheretherketone wafer and polish it successively with 600#, 800#, 1000#, 1200# and 1500# silicon sandpapers. The specification of the polyetheretherketone wafer can be 10 mm × 10 mm × 1 mm.

[0058] (2) The polished polyetheretherketone wafer is ultrasonically washed successively with propanol, absolute ethanol and deionized water for 30 min, and then dried in an oven at 50 °C for standby;

[0059] (3) Immerse the dried polyetheretherketone in concentrated sulfuric acid at room temperature, take out the acidified polyetheretherketone after 6 min, and then ultrasonically oscillate the acidified polyetheretherketone successively in acetone, absolute ethanol and deionized water for 10 min to remove the remaining sulfuric acid on the surface of the sample. Then air dry it to obtain sulfonated polyetheretherketone.

[0060] The modified polyetheretherketone can be obtained by immersing the above-prepared sulfonated polyetheretherketone in a dopamine solution, carrying out a light-shielded reaction in a constant-temperature shaker at a temperature of 37 °C and a rotation speed of 60 rpm / min for 24 h, then taking out the sample for ultrasonic washing with deionized water for 5 min, and air drying it, thus obtaining the modified polyetheretherketone. Through the above method, it is possible to form a polydopamine coating in the form of self-assembly by dopamine, and make the polydopamine coating firmly adhere to the surface of polyetheretherketone.

[0061] Among them, the dopamine solution can be a 10 mM Tris-HCl buffer solution with a pH of 8.5 and a dopamine content of 2 g / L.

[0062] It can be understood that the polydopamine coating can effectively reduce the water contact angle of sulfonated polyetheretherketone and improve the hydrophilicity of the prepared modified polyetheretherketone.

[0063] Example 2

[0064] This embodiment also provides an artificial bone composite material, which can be prepared through the following method. Specifically, the preparation method of the artificial bone composite material in this embodiment can be referred to Figure 1 as shown, and it includes the following steps:

[0065] (Ⅰ) Preparation of Collagen-Hydroxyapatite

[0066] Dissolve 1.1 g of collagen in 0.2 mol / L acetic acid solution to prepare a 1.1 mg / mL collagen solution. Add 50 mL of 0.2 mol / L CaCl2 solution to 1000 mL of 1.1 mg / mL collagen solution under stirring conditions. Then slowly add 50 mL of 1.2 mol / L NaH2PO4 solution to the above-mentioned collagen solution to obtain Mixture 1. Among them, the molar ratio of calcium ions to phosphate ions in Mixture 1 is 1.67.

[0067] Add NaOH solution to Mixture 1 to adjust the pH of Mixture 1 to 7.4, then stir for 72 h, and centrifuge to collect the white gel, which is collagen-hydroxyapatite. Wash the white gel with ultrapure water three times to remove the soluble salts on the white gel.

[0068] (Ⅱ) Preparation of Poly(lactic acid)-Collagen-Hydroxyapatite

[0069] Take 2.2 g of the collagen-hydroxyapatite prepared in step (Ⅰ), disperse it fully in 50 mL of distilled water, and perform ultrasonic treatment for 30 min to obtain Colloid 1.

[0070] Then add Colloid 1 to a dichloromethane solution with a volume of 176 mL and containing L-lactic acid to obtain Mixture 2. The mass concentration of L-lactic acid in the dichloromethane solution is 4%.

[0071] Stir Mixture 2 with a high-speed shear emulsifier at 2500 r / min, and at the same time add a large amount of ethanol until the precipitate in Mixture 2 is completely precipitated. Then remove the clear liquid in Mixture 2 to obtain the precipitate.

[0072] Wash and separate the obtained precipitate to form a uniform and viscous slurry. Then inject the slurry into a polytetrafluoroethylene cylindrical mold with a diameter of 1.2 cm and a height of 2 cm, and the height of the added slurry is 1 cm. Perform vacuum degassing in the cylindrical mold, pre-cool overnight at -20°C to -60°C for 12 h, and then freeze-dry at -20°C to -60°C for 36 h.

[0073] Soak the freeze-dried sample in a 95% ethanol solution containing 1 mol / L EDC and 0.25 mol / L NHS for cross-linking, wash with deionized water, and then perform freeze-drying to obtain the artificial bone composite material of Example 2. Its specific structure can be referred to Figure 2 as shown.

[0074] Among them, the hydroxyapatite in this embodiment is in-situ mineralized at the nucleation sites of carbonyl and carboxyl groups in collagen. Under the regulation of the collagen solution, poly-L-lactic acid can form hydrogen bonds with collagen, improving the interfacial properties. The regulation of collagen significantly improves the mechanical properties of poly-lactic acid - collagen - hydroxyapatite (i.e., artificial bone composite material). The compressive strength of the artificial bone composite material reaches 13.4 MPa, which is 22.9% higher than that of poly-lactic acid - hydroxyapatite. The elastic modulus of poly-lactic acid - collagen - hydroxyapatite (i.e., artificial bone composite material) is 84.96 MPa, which is 26% higher than that of poly-lactic acid - hydroxyapatite, meeting the requirements of bone tissue repair materials. The regulation of collagen also reduces the crystallization of the artificial bone composite material, extends the degradation period, and continuously releases Ca during the degradation process. 2+ .

[0075] In this embodiment, the poly-lactic acid can be poly-L-lactic acid. Poly-L-lactic acid (PLLA) has a relatively high crystallinity, so its mechanical properties, heat resistance are relatively good, and it is more rigid, suitable for occasions with higher requirements for material properties. Due to its relatively high crystallinity, the glass transition temperature (Tg) of PLLA is also relatively high, and the melting point is higher. PLLA is mainly applied in the medical and biomedical fields, such as for sutures, implants, drug carriers, etc., because it has relatively high crystallinity, mechanical strength and heat resistance. ;

[0076] And in the preparation method, the artificial bone composite material is immersed in 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (i.e., EDC / NHS) for crosslinking after freeze-drying. This test method enables the sample to be crosslinked after freeze-drying and forming, and can be crosslinked evenly, thereby further improving the compressive strength of the prepared artificial bone composite material.

[0077] Example 3

[0078] This embodiment provides a second type of artificial bone composite material, which can be prepared in the following manner. Compared with Example 2, in the preparation process, the calcium source is changed from CaCl2 solution in Example 2 to Ca(NO3)2, and NaH2PO4 in Example 2 is changed to Na2HPO4. Specifically, the preparation method of the artificial bone composite material in this embodiment includes the following steps:

[0079] (11) Preparation of collagen-hydroxyapatite

[0080] Under stirring conditions, 0.25 g of collagen is dissolved in 260 mL of acetic acid solution with a concentration of 1% W / V. After the collagen is completely dissolved, it is diluted with distilled water to obtain a collagen solution with a mass concentration of 5 mg / mL.

[0081] Add 2.5 g of Ca(NO3)2 to the collagen solution. After it is dissolved, add 0.03 mol / L Na2HPO4 dropwise to the above solution to obtain a first mixed solution. Among them, the amount of Na2HPO4 added is such that the molar ratio of calcium ions to phosphate ions in the first mixed solution is 1.67. Then, the pH of the first mixed solution can be maintained at 10 by ammonia water and stirred for 3 hours. After standing and aging at room temperature for at least 24 h, filter the first mixed solution to obtain a precipitate, and freeze-dry the precipitate (the precipitate here is the collagen-hydroxyapatite in Example 3).

[0082] (12) Preparation of polylactic acid - collagen - hydroxyapatite

[0083] Take 2.2 g of the collagen-hydroxyapatite prepared in step (11), disperse it fully in 50 mL of distilled water, and ultrasonically treat it for 30 min to obtain a first colloid.

[0084] Then add the first colloid to a dichloromethane solution with a volume of 176 mL and containing poly(L-lactic acid). The mass concentration of poly(L-lactic acid) in the dichloromethane solution is 4%.

[0085] Then stir the second mixed solution with a high-speed shear emulsifier at 2500 r / min, and at the same time add a large amount of ethanol until the precipitate in the second mixed solution precipitates completely. Then remove the clear liquid in the second mixed solution to obtain a precipitate.

[0086] Wash and separate the obtained precipitate to form a uniform and viscous slurry. Then inject the slurry into a cylindrical mold made of polytetrafluoroethylene with a diameter of 1.2 cm and a height of 2 cm. The height of the added slurry is 1 cm. Vacuum degas in the cylindrical mold, pre-cool at -20°C to -60°C overnight for 12 h, and then freeze-dry at -20°C to -60°C for 36 h.

[0087] Then soak the freeze-dried sample in a 95% ethanol solution containing 1 mol / L EDC and 0.25 mol / L NHS for crosslinking, wash with deionized water, and then freeze-dry to obtain the artificial bone composite material of Example 3.

[0088] It can be understood that in this example, Ca(NO3)2 can be prepared in the following way: Weigh 2.25 g of Ca(OH)2, dilute it with 10 mL of water, and stir for standby. Then use a pipette to take 5 mL of HNO3 with a concentration of 65% - 68%, and after adding 15 mL of water for dilution. Then take 17.1 mL of the HNO3 solution and drop it into the Ca(OH)2 solution to obtain a Ca(NO3)2 solution with a pH of 7 and a concentration of 185 mg / mL.

[0089] Example 4

[0090] The raw materials and preparation process of the artificial bone composite material provided in Example 4 are the same as those in Example 2, except that the volume ratio of the collagen solution to the CaCl2 solution in Example 4 is 15:1.

[0091] Comparative Example 1

[0092] This comparative example provides an artificial bone composite material. Compared with Example 2, the raw materials in this comparative example lack collagen and poly(L-lactic acid). Therefore, the preparation method of the artificial bone composite material provided in this comparative example includes the following steps:

[0093] Weigh 1.11 g of CaCl2 and dissolve it in 50 mL of water to obtain a CaCl2 solution. Under stirring conditions, add the obtained CaCl2 solution to 1 L of deionized water and stir for 15 minutes to obtain a diluted CaCl2 solution.

[0094] Dissolve 0.72 g of NaH2PO4 in 50 mL of water respectively to obtain a NaH2PO4 solution. Slowly add the NaH2PO4 solution to the diluted CaCl2 solution to obtain a mixture I. Then adjust the pH value of the mixture I to 7.5 with 0.1 M NaOH. Stir the mixture I after adjusting the pH at room temperature at a rotation speed of 200 rpm for 72 hours. Then centrifuge at 5000 rpm for 10 minutes to collect the solid, wash the precipitate twice with deionized water, and freeze-dry to obtain the solid, and the obtained solid is the artificial bone composite material prepared in Comparative Example 1.

[0095] Comparative Example 2

[0096] This comparative example provides an artificial bone composite material. Compared with Example 2, the raw materials in this comparative example lack poly(L-lactic acid). Therefore, the preparation method of the artificial bone composite material provided in this comparative example includes the following steps:

[0097] Dissolve 11 mg of collagen in 10 mL of 0.2 mol / L acetic acid solution and prepare a 1.1 mg / mL collagen solution.

[0098] Under stirring conditions, add 1 mL of 0.2 mol / L CaCl2 solution to 9 mL of 1.1 mg / mL collagen solution. Then slowly add 0.6 mL of 0.12 mol / L NaH2PO4 solution to the above collagen solution to obtain a mixture I, so that the molar ratio of calcium ions to phosphate ions in the mixture I is 1.67.

[0099] Then, add NaOH solution to the first mixture to adjust the pH of the first mixture to 7.4. After stirring for 72 h, centrifuge to collect the white jelly. Then wash the white jelly with ultrapure water three times to remove the soluble salts on the white jelly, and the artificial bone composite material of Comparative Example 2 is obtained. Its specific structure can be referred to Figure 3 as shown.

[0100] Performance Test

[0101] In order to verify the performance of the artificial bone composite material provided in this example, the technical personnel designed the following test and verification experiments.

[0102] (1) Take the performance of the artificial bone composite material prepared in Example 2 as an example for the following research.

[0103] 1.1 Infrared spectrum

[0104] Observe the infrared spectrum of the artificial bone composite material prepared in Example 2 through a Fourier transform infrared spectrometer to obtain Figure 4 . By analyzing Figure 4 , it can be known that: the FT-IR spectrum of the artificial bone composite material shows vibration peaks at 1640, 1031, 563, and 604 cm -1 . Among them, the vibration peak at 1640 cm -1 belongs to the C=O bond, while the vibration peaks at 1031, 563, and 604 cm -1 belong to phosphate groups, which further indicates that the artificial bone composite material contains collagen and hydroxyapatite.

[0105] 1.2 Compressive strength

[0106] Place the artificial bone composite material prepared in Example 2 under a compressive strength testing machine to test its compressive strength, and obtain Figure 5 and Figure 6 . Among them, Figure 5 the left figure in it is a schematic diagram of the artificial bone composite material of Example 2 placed under the compressive strength testing machine; Figure 5 the right figure in it is a schematic diagram of the artificial bone composite material in Example 2 being crushed by the compressive strength testing machine. Figure 6 is the relationship curve between the displacement in the artificial bone composite material of Example 2 and the pressure applied by the compressive strength testing machine. By analyzing Figure 6 , it can be known that: using the compression test to evaluate the mechanical properties of the artificial bone composite material, the results are as Figure 6 shown. The artificial bone composite material has a very high compressive strength and good mechanical strength.

[0107] 1.3 X-ray diffraction technology

[0108] The crystal type of the artificial bone composite material of Example 2 was characterized using an X-ray diffractometer. The XRD analysis results are as Figure 7 shown. By Figure 7 it can be seen that Figure 7 there are characteristic diffraction peaks at 25.5°, 31.9°, 39.2°, 46.4° and 48.9° in , corresponding to the diffraction planes of hydroxyapatite (002), (211), (310), (222) and (213) respectively. This indicates that nano-hydroxyapatite was successfully synthesized using collagen as a template.

[0109] 1.4 Thermogravimetric analysis technology

[0110] The content of organic and inorganic substances in the artificial bone composite material prepared in Example 2 was evaluated using thermogravimetric analysis technology (TGA), and Figure 8 . By analyzing Figure 8 it can be seen that Figure 8 the TGA curve in shows two weight loss stages: in the first stage when the temperature is below 200 °C, it corresponds to the evaporation of water in the artificial bone composite material. In the second stage when the temperature is between 200 and 600 °C, it belongs to the thermal decomposition of the organic components in the artificial bone composite material. When the temperature rises above 600 °C, there is an obvious weight loss phenomenon in the artificial bone composite material, indicating the thermal decomposition of polylactic acid. When the temperature rises above 800 °C, there is no obvious weight loss phenomenon in the artificial bone composite material, indicating that the remaining mass is stable inorganic material. And it was measured that the content of hydroxyapatite in the artificial bone composite material is 40%. This indicates that the content of organic and inorganic substances in the artificial bone composite material prepared in Example 2 is similar to that of natural bone, thus indicating that the artificial bone composite material prepared in Example 2 meets the requirements of artificial bone.

[0111] (2) The following verification analysis was carried out on the modified polyetheretherketone

[0112] 2.1 The components of polyetheretherketone, sulfonated polyetheretherketone and modified polyetheretherketone were detected using an X-ray photoelectron spectrometer (XPS)

[0113] The polyetheretherketone in Example 1, the prepared sulfonated polyetheretherketone and modified polyetheretherketone were respectively placed in an X-ray photoelectron spectroscopy (XPS) for analysis, and Figure 9 .

[0114] Among them, Figure 9 Figure (a) in is the main element composition of polyetheretherketone. Figure 9 Figure (b) in is the main element composition of sulfonated polyetheretherketone. Figure 9 Figure (c) in is the main element composition of modified polyetheretherketone. By Figure 9Analysis of (a), (b), and (c) shows that the polyether ether ketone material mainly contains C and O elements. The main elemental components of sulfonated polyether ether ketone are still C and O. In addition, a very weak S element peak can be detected, indicating that sulfonated polyether ether ketone can basically remove the S element remaining on its surface after sufficient ultrasonic washing. The main component of polydopamine is N element. An obvious N element peak is detected on the surface of the modified polyether ether ketone modified by the polydopamine coating, indicating that polydopamine has successfully adhered to the surface of the polyether ether ketone material. Thus, it can be proved that the modified polyether ether ketone prepared in Example 1 is modified by the polydopamine coating.

[0115] 2.2 Verification of the bioactivity of the modified polyether ether ketone

[0116] 2.2.1 Observation of cell morphology on different material surfaces

[0117] After co-culturing the polyether ether ketone, sulfonated polyether ether ketone, and modified polyether ether ketone in Example 1 with MC3T3-E1 cells for 12 h respectively, the cell adhesion morphology maps of the surfaces of the above materials were obtained by scanning electron microscopy, as Figure 10 shown.

[0118] Among them Figure 10 Figure (d) is the cell adhesion morphology map of the polyether ether ketone surface; Figure 10 Figure (e) is the cell adhesion morphology map of the sulfonated polyether ether ketone surface; Figure 10 Figure (f) is the cell adhesion morphology map of the modified polyether ether ketone surface.

[0119] Through the analysis of Figure 10 , it can be seen that for the cell adhesion morphology map of the polyether ether ketone surface, there are fewer cells on its surface, and the cell shape is mostly flat spherical, with the cell morphology slightly extended and starting to extend pseudopodia around. For the cell adhesion map of the sulfonated polyether ether ketone surface, compared with the cells on the polyether ether ketone surface, the cell morphology has changed, and the contact area between the cells on the sulfonated polyether ether ketone surface and the polyether ether ketone surface has increased. The cells are imaged in an irregular flat shape, and the cells have extended pseudopodia on the sulfonated polyether ether ketone surface. For the modified polyether ether ketone, the number of cells on the surface of the modified polyether ether ketone is the largest, and the cell spreading is the most obvious. The cells on the surface of the modified polyether ether ketone adhere to its surface in a spindle shape and a flat shape, and the cells extend more pseudopodia. Through the above analysis, it can be seen that sulfonated polyether ether ketone and modified polyether ether ketone can promote the adhesion and spreading of early cells on the surface of the polyether ether ketone material to a certain extent.

[0120] 2.2.2 Cell proliferation results

[0121] After polyetheretherketone (PEEK), sulfonated polyetheretherketone (SPEEK), and modified polyetheretherketone (mPEEK) were co-cultured with MC3T3-E1 cells for 1 day, 2 days, and 3 days respectively, the cell proliferation on the surfaces of the above three materials was as follows Figure 11 shown. By analyzing Figure 11 , it can be seen that when the cells were cultured on the above three materials for 1 day, the number of cells on the surface of SPEEK was slightly higher than that of the other two materials, and there was no significant difference in the number of cells among the three materials (P > 0.05). When the cells were cultured on the above three materials for 2 and 3 days respectively, the cell proliferation activities of SPEEK and mPEEK were higher than that of PEEK, and the differences in cell proliferation activities on the surfaces of the three materials were statistically significant (P < 0.05). Moreover, the absorbance values on the surfaces of the three materials showed an upward trend with the extension of the co-culture time with cells. Thus, it can be proved that the above three materials are non-toxic to cells and have good biocompatibility.

[0122] (3) Test and analyze the properties of the artificial bone composite materials prepared in Examples 1 to 3, Comparative Example 1, and Comparative Example 2

[0123] 3.1 Contact Angle Detection (Contact Angle, CA)

[0124] The contact angles of the artificial bone composite materials prepared in Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Example 2 were detected respectively. The operation of contact angle detection can be as follows: At room temperature, a droplet imaging analysis system was used to measure the surface wettability of the above 5 artificial bone composite materials. A syringe can be used to suck 10 μL of deionized water solution and vertically drop it onto different sites on the surface of the artificial bone composite material. Five parallel data were taken for each artificial bone composite material, and the average value of the data was calculated. The shape of the water droplet was captured by the imaging system to obtain the data in Table 1 below.

[0125] Contact Angle Example 1 25.6°±1.23° Example 2 35.3°±3.51° Example 3 45.2°±2.18° Comparative Example 1 109.6°±1.56° Comparative Example 2 88.0°±3.16°

[0126] By analyzing the data of the water contact angles on the surfaces of the above five artificial bone composites, it can be seen that the artificial bone composite prepared in Example 1 has the smallest contact angle, indicating that it has the best hydrophilicity. Compared with the artificial bone composite prepared in Example 1, the artificial bone composite prepared in Example 2 does not contain modified polyetheretherketone in its raw materials. Thus, it can be known that adding modified polyetheretherketone to the artificial bone composite can effectively improve the hydrophilicity of the prepared artificial bone composite. And the better the hydrophilicity of the artificial bone composite, the more easily it can absorb the surrounding moisture after being implanted into the human body, forming a thin water film, which makes it easier for bone cells in the body to attach, thereby promoting bone growth. Moreover, by comparing Comparative Example 2 with Example 2, it can be seen that the hydrophilicity of the artificial bone composite prepared in Example 2 is much better than that of the artificial bone composite prepared in Comparative Example 2. The preparation processes of the artificial bone composites in Comparative Example 2 and Example 2 are the same, and the difference is that the raw materials of the artificial bone composite in Comparative Example 2 do not contain left-handed polylactic acid. Thus, by comparing Comparative Example 2 with Example 2, it can be known that left-handed polylactic acid can effectively improve the hydrophilicity of the prepared artificial bone composite.

[0127] By comparing Comparative Example 1 with Comparative Example 2, it can be seen that the raw materials of the artificial bone composite in Comparative Example 1 do not contain collagen. And the contact angle of the artificial bone composite in Comparative Example 2 is smaller than that of the artificial bone composite in Comparative Example 1. Thus, it can be known that adding collagen to the artificial bone composite can also effectively improve the hydrophilicity of the prepared artificial bone composite.

[0128] 3.2 SEM Scanning Electron Microscope

[0129] The artificial bone composite prepared in Comparative Example 2 was observed under a SEM scanning electron microscope, and the results are as Figure 12 . Figure 12 The left and right figures in the following are the electron microscope images of the artificial bone composite in Comparative Example 2 at different magnification diameters. Through Figure 12 it can be known that the surface of the artificial bone composite prepared in Comparative Example 2 is not dense, resulting in its weak compressive strength. Thus, it can be proved that adding collagen to the raw materials of the artificial bone composite can enhance the compressive strength of the prepared artificial bone composite.

[0130] The basic principles, main features and advantages of the present invention have been described above. Those skilled in the art of this industry should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification is only the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an artificial bone composite material, characterized in that, It includes the following steps: Adding CaCl2 solution and NaH2PO4 solution to the collagen solution in proportion, to obtain a mixed solution 1; the molar ratio of calcium ions to phosphate ions in the mixed solution 1 is 1.67; Then, adding alkali solution to the mixed solution 1 to adjust the pH of the mixed solution 1 to 7.4, mixing evenly and collecting white colloid by centrifugation; The white colloid is fully dispersed in distilled water and subjected to ultrasonic treatment to obtain colloid 1; the colloid 1 and the modified polyetheretherketone powder are respectively added to a dichloromethane solution containing L-polylactic acid in proportion, and mixed evenly by high-speed shearing to obtain a mixed solution 2; Adding ethanol solution to the mixed solution 2 until the precipitate in the mixed solution 2 is completely precipitated; removing the clear liquid in the mixed solution 2 to obtain the precipitate, and washing and separating the precipitate to form a uniform, viscous slurry; The slurry is injected into a mold for vacuum degassing, and freeze-dried at -20°C to -60°C to obtain an intermediate 1; The intermediate is immersed in a 95% ethanol solution containing 1 mol / L EDC and 0.25 mol / L NHS for cross-linking, washed with deionized water, and freeze-dried to obtain the artificial bone composite material.

2. The preparation method of the artificial bone composite material according to claim 1, characterized in that, The modified polyetheretherketone is prepared by the following steps: preparing sulfonated polyetheretherketone by sulfuric acid chemical etching, and then immersing the sulfonated polyetheretherketone in a dopamine solution to react in the dark, thereby obtaining the modified polyetheretherketone.

3. The preparation method of the artificial bone composite material according to claim 2, characterized in that, The modified polyetheretherketone powder is obtained by grinding the modified polyetheretherketone into particles with a particle size of 0.5nm to 1nm using an ultrafine grinder.

4. The preparation method of the artificial bone composite material according to claim 2, characterized in that, The preparation method of the sulfonated polyetheretherketone is as follows: (1) Prepare a polyetheretherketone disc and grind and polish it with 600#, 800#, 1000#, 1200# and 1500# silica sandpaper in sequence; (2) The polished polyetheretherketone disc is ultrasonically cleaned with propanol, anhydrous ethanol and deionized water in sequence, and then dried at 50° C. for later use; (3) The dried polyetheretherketone is immersed in concentrated sulfuric acid at room temperature, and the acidified polyetheretherketone is taken out after 6 minutes. The acidified polyetheretherketone is then placed in acetone, anhydrous ethanol and deionized water for ultrasonic vibration in sequence, and then air-dried to obtain the sulfonated polyetheretherketone.

5. The preparation method of the artificial bone composite material according to claim 2, characterized in that, The dopamine solution is a 10 mM Tris-HCl buffer solution with a pH of 8.5 and a dopamine content of 2 g / L.

6. The preparation method of the artificial bone composite material according to claim 1, wherein, The concentration of the CaCl2 solution is 0.2 mol / L; the concentration of the collagen solution is 1.1 mg / mL, and the volume ratio of the collagen solution to the CaCl2 solution is 20:1 to 15:1; the concentration of the NaH2PO4 is 1 mol to 1.5 mol / L, and the volume ratio of the NaH2PO4 to the CaCl2 solution is 1:

1.

7. The preparation method of the artificial bone composite material according to claim 1, wherein, The mold is a cylindrical mold made of polytetrafluoroethylene with a diameter of 1.2 cm and a height of 2 cm; the height of the slurry added into the mold is 1 cm.

8. The preparation method of the artificial bone composite material according to claim 1, wherein, The collagen is type I bovine collagen; the mass concentration of poly(L-lactic acid) in the dichloromethane solution containing poly(L-lactic acid) is 4% - 5%, and the volume ratio of the dichloromethane solution to the first colloid is 1:3 - 1:4; And / or, the lye is any one of NaOH or Ca(OH)2.

9. The preparation method of the artificial bone composite material according to claim 8, wherein, The collagen solution is prepared by dissolving type I bovine collagen in an acetic acid solution with a concentration of 0.2 mol / L; and the solid-liquid ratio of the type I bovine collagen to the acetic acid solution is 10 mg:1 mL.

10. An artificial bone composite material, characterized in that, It is prepared by using the preparation method of the artificial bone composite material described in any one of claims 1 - 9; the compressive strength of the artificial bone composite material is greater than or equal to 13.4 MPa; the water contact angle on the surface of the artificial bone composite material is 25.6° ± 1.23°.

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