Preparation method of an injectable hyaluronic acid hydrogel containing a BMP2 mimetic peptide and SDF-1

Abstract

The present invention discloses a preparation method of an injectable hyaluronic acid hydrogel containing a BMP2 mimetic peptide and SDF-1, which is characterized in that: 1) preparing a hyaluronic acid hydrogel precursor solution containing the BMP2 mimetic peptide; 2) preparing silk fibroin microspheres compounded with SDF-1: 2-1) preparing the silk fibroin microspheres; 2-2) compounding SDF-1 with the silk fibroin microspheres in a solution through the principle of positive and negative charge adsorption to form silk fibroin microspheres compounded with SDF-1; 3) mixing the silk fibroin microspheres compounded with SDF-1 with the hyaluronic acid hydrogel precursor solution, and an injectable hyaluronic acid hydrogel containing the BMP2 mimetic peptide and SDF-1 can be formed at room temperature. The present invention uses hyaluronic acid as the main body of the hydrogel, introduces silk fibroin microspheres to compound SDF-1, and simultaneously encapsulates the BMP2 mimetic peptide, solves the problems of rapid release in vivo of SDF-1 and BMP2 factors and the immunogenicity problem of the BMP2 factor, and successfully prepares an injectable hyaluronic acid hydrogel modified with the BMP2 mimetic peptide and encapsulating the compounded SDF-1 silk fibroin microspheres, laying a foundation for promoting bone tissue regeneration in vivo with this biomaterial.

Description

Technical Field

[0001] The present invention relates to the field of materials, and particularly to a preparation method of an injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1. Background Art

[0002] Hydrogel is a special soft and wet material with a three-dimensional network structure crosslinked by chemical bonds, hydrogen bonds, van der Waals forces or physical entanglement, etc. It can swell but not dissolve in water or biological body fluids, and has both solid and liquid properties. This characteristic makes it widely used in biomedical fields such as tissue engineering and drug carriers. Since the three-dimensional network structure of the hydrogel is similar to the natural extracellular matrix, the hydrogel material can provide a supporting role and a metabolic space for cell adhesion, proliferation and growth. The water absorption and swelling characteristics of the hydrogel and its permeation and diffusion of small molecules endow it with good biocompatibility, and the good flexibility and high elasticity of the hydrogel can greatly reduce the damage to cells. Therefore, it can be widely used in fields such as tissue engineering repair.

[0003] Stromal cell-derived factor (SDF-1) is a super cell-derived factor that can recruit stem cells and cause the recruitment behavior of stem cells. At the same time, studies have shown that SDF-1 can be secreted on the surface of periodontal ligament stem cells, and similar to vascular endothelial growth factor and fibroblast growth factor 2 (FGF2), it has the potential to support and promote the differentiation of periodontal ligament stem cells. In addition, SDF-1 participates in the regulation of angiogenesis and plays an important role during embryogenesis, development and tissue regeneration, partly by recruiting endothelial progenitor cells and guiding the transport of stem cells, and can migrate periodontal ligament stem cells to the periodontal tissue defect site by activating lipid phosphokinase (P13K), and at the same time can regulate periodontal tissue inflammation and regulate the immune response. At present, the use of SDF-1 factor focuses on single injection or soaking biological materials in SDF-1 diluent, which is not conducive to the sustained release and continuous action of SDF-1 factor.

[0004] BMP2 (bone morphogenetic protein 2) mimetic peptide (sequence: KIPKASSVPTELSAISTLYL) has a biological role similar to BMP2 and can promote osteogenesis of stem cells. However, currently, high doses of BMP2 are instead not conducive to the osteogenic differentiation of stem cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method of an injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1. The preparation method is simple, and solves the problem of rapid release in vivo involved in the single use of SDF-1 and BMP2 factors.

[0006] The technical solution adopted by the present invention is as follows: A preparation method of an injectable hyaluronic acid hydrogel containing BMP2 and SDF-1, comprising the following steps:

[0007] 1) Prepare a hyaluronic acid hydrogel precursor solution containing a BMP2 mimetic peptide

[0008] 1-1) Prepare adipic dihydrazide-modified hyaluronic acid HA-ADH;

[0009] 1-2) Prepare oxidized hyaluronic acid HA-CHO;

[0010] 1-3) The products HA-ADH and HA-CHO prepared above are respectively dissolved with a BMP2 mimetic peptide solution to form a HA-ADH solution containing a BMP2 mimetic peptide and a HA-CHO solution containing a BMP2 mimetic peptide. Both are the hyaluronic acid hydrogel precursor solutions containing a BMP2 mimetic peptide;

[0011] 2) Prepare silk fibroin microspheres composite with SDF-1

[0012] 2-1) Prepare silk fibroin microspheres: Using Ca 2 CO 3 as the core of the silk fibroin microspheres, silk fibroin is coated outside Ca 2 CO 3 ;

[0013] 2-2) Composite SDF-1 with the silk fibroin microspheres in solution through the principle of positive and negative charge adsorption to form silk fibroin microspheres composite with SDF-1;

[0014] 3) Mix the silk fibroin microspheres composite with SDF-1 with the hyaluronic acid hydrogel precursor solution containing a BMP2 mimetic peptide to form an injectable hyaluronic acid hydrogel containing a BMP2 mimetic peptide and SDF-1.

[0015] Preferably, the specific preparation method of the adipic dihydrazide-modified hyaluronic acid HA-ADH is: React hyaluronic acid with adipic dihydrazide under the activation conditions of EDC and HOBT to prepare HA-ADH.

[0016] Preferably, the specific preparation method of the oxidized hyaluronic acid (HA-CHO) is: Oxidize hyaluronic acid with periodic acid to prepare HA-CHO.

[0017] Preferably, the specific preparation method of the silk fibroin microspheres is:

[0018] a. Prepare the core of the silk fibroin microspheres - Ca 2 CO 3 template: Mix Na 2 CO 3The solution is added to the stirred CaCl 2 in the solution to form Ca 2 CO 3 template;

[0019] b. Centrifuge the Ca 2 CO 3 template prepared in step a, discard the supernatant, add ethanol-water, centrifuge, discard the supernatant, and then add water and centrifuge multiple times, discarding the supernatant;

[0020] c. Add water to resuspend, then add purified silk fibroin. After vertical suspension, centrifuge and wash, add methanol-water, vertically suspend again, and centrifuge and wash.

[0021] d. Repeat step c for multi-layer coating;

[0022] e. When the desired number of layers is wrapped, add glutaraldehyde solution, vertically suspend, centrifuge, and wash with water to obtain silk fibroin microspheres;

[0023] Preferably, in step a, the molar ratio of Na 2 CO 3 to CaCl 2 CO 3 in the preparation of the Ca 2 template is 1:1.

[0024] Preferably, in step b, the volume ratio of ethanol to water added is 1:1 - 1:3.

[0025] Preferably, in step c, the volume ratio of methanol to water added is 1:5 - 1:10.

[0026] Preferably, in step c, the specific preparation process of the purified silk fibroin is: add LiBr solution to silk fibroin powder, stir in a preheated water bath, then transfer to a dialysis bag and dialyze with ultrapure water, and then centrifuge and filter through a membrane for storage.

[0027] Further preferably, the cut-off molecular weight of the dialysis bag is 3500 MW.

[0028] Further preferably, the pore size of the membrane is 0.22 μm.

[0029] The beneficial effects of the present invention are as follows: in the preparation method of the injectable hyaluronic acid hydrogel disclosed in the present invention, SDF-1 is compounded with silk fibroin microspheres, and the BMP2 mimetic polypeptide is introduced into the HA hydrogel at the same time, and then the silk fibroin microspheres compounded with SDF-1 are mixed with the HA hydrogel precursor solution containing the BMP2 mimetic polypeptide to form an injectable hyaluronic acid hydrogel containing the BMP2 mimetic polypeptide and the SDF-1 silk fibroin microspheres. The preparation method is simple, and the adsorption effect of silk fibroin on SDF-1 and the encapsulation effect of the hydrogel on the BMP2 mimetic polypeptide solve the in vivo burst release problem involved in the use of SDF-1 and BMP2 factors alone. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solution of the present invention, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1 This is the H NMR spectrum of hyaluronic acid (HA);

[0032] Figure 2 This is the H NMR spectrum of hyaluronic acid (HA-ADH) modified with adipic acid dihydrazide. The methylene signal peak at δ=2.1-2.3ppm confirms that adipic acid dihydrazide successfully modifies hyaluronic acid. Using N-acetylmethyl (δ=1.91-1.92ppm) as the internal standard, the degree of substitution is calculated to be 30.78%. ;

[0033] Figure 3 Infrared spectra of HA, HA-CHO and HA-ADH, 1785cm -1 The absorption peak of aldehyde group is 1579cm -1 The absorption peak is the amide bond;

[0034] Figure 4 This is a scanning electron microscope image of silk fibroin microspheres;

[0035] Figure 5 This is the Zata potential diagram of silk fibroin and composite SDF-1 silk fibroin microspheres;

[0036] Figure 6 is a scanning electron micrograph of a hyaluronic acid hydrogel containing a BMP2 mimetic peptide (excluding silk fibroin microspheres, i.e., formed by mixing a HA-CHO-BP solution with a HA-ADH-BP solution);

[0037] Figure 7Scanning electron micrograph of a hyaluronic acid hydrogel containing a BMP2 mimetic peptide and an SDF-1 silk fibroin microsphere (including the silk fibroin microsphere, that is, the silk fibroin microsphere complexed with SDF-1 is mixed with the HA-CHO-BP solution and the HA-ADH-BP solution). Detailed implementation manners

[0038] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention and the corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. The technical solutions provided by the embodiments of the present invention are described in detail below with reference to the drawings.

[0039] Example 1

[0040] I. Preparation of a hyaluronic acid hydrogel precursor solution containing a BMP2 mimetic peptide

[0041] 1. Preparation of hyaluronic acid adipic dihydrazide (HA-ADH)

[0042] 1. Preparation of hyaluronic acid modified with adipic dihydrazide (HA-ADH)

[0043] Take 200 mg (90 kDa) of hyaluronic acid (HA) and dissolve it in 40 mL of distilled water. Add 2.6 g of adipic dihydrazide and react for 30 min. Weigh 0.31 g of EDC and 0.306 g of HOBT, and dissolve them thoroughly in 40 mL of DMSO (ultrapure water:DMSO = 1:1). After complete dissolution, slowly add the DMSO to the HA solution. React for 4 h, and adjust the pH to 6.8 every 30 min. After the 4-h reaction, continue to stir the reaction at room temperature for 24 h. Transfer the solution to a dialysis bag and dialyze for 3 days, filter with a 0.22-μm filter membrane, and freeze-dry to obtain solid HA-ADH. 1 The degree of substitution of adipic dihydrazide was detected by 1H-NMR, and the calculated value was 30.78%. The 1H-NMR detections of HA and HA-ADH are as 1 shown in Figure 1 and Figure 2 shown.

[0044] 2. Preparation of oxidized hyaluronic acid (HA-CHO)

[0045] Dissolve 200 mg (90 kDa) of hyaluronic acid in 20 mL of distilled water until fully dissolved. Add 103 mg of sodium periodate for oxidation and react for 2 h. Then add an excess of ethylene glycol to terminate the reaction. Transfer the solution to a dialysis bag and dialyze for 3 days. Filter through a 0.22 μm filter membrane and lyophilize to obtain solid HA-CHO.

[0046] Dissolve 50 mg of HA-CHO in 20 mL of 0.25 mol / L hydroxylamine hydrochloride solution. After reacting for 5 h, determine the oxidation degree of HA-CHO by potentiometric titration, and the measured value is 26.32%.

[0047] Use Fourier transform infrared spectroscopy to determine the formation of aldehyde groups in oxidized HA. Take a small amount of freeze-dried HA-CHO, HA-ADH, and HA, and perform tests by the transmission method. The spectral range of infrared testing is 400 cm -1 -4000 cm -1 ,and the infrared spectrum detection is as Figure 3 shown.

[0048] 3. Preparation of a hyaluronic acid hydrogel precursor solution containing BMP2 mimetic peptide

[0049] HA-ADH and HA-CHO are respectively dissolved in a BMP2 mimetic polypeptide solution with a concentration of 2 mg / mL to form 3 wt% solutions, preparing an HA-ADH solution containing BMP2 mimetic peptide (HA-CHO-BP solution) and an HA-CHO solution containing BMP2 mimetic peptide (HA-ADH-BP solution). These two are the hyaluronic acid hydrogel precursor solutions containing BMP2 mimetic peptide (the hyaluronic acid hydrogel precursor solution containing BMP2 mimetic peptide refers to the independent and unmixed HA-CHO-BP solution and HA-ADH-BP solution. When mixed with silk fibroin microspheres complexed with SDF-1, the HA-CHO-BP solution and HA-ADH-BP solution are mixed to form an HA hydrogel containing BMP2 mimetic peptide).

[0050] II. Preparation of silk fibroin microcapsule microspheres complexed with SDF-1

[0051] 1. Purification of silk fibroin

[0052] Weigh 4 g of silk fibroin powder into a 50 mL round-bottom flask, add 20 mL of 9.3 M LiBr solution, place it in a water bath preheated to 60 °C and stir for 4 h. Then transfer it to a 3500 MW dialysis bag and dialyze with ultrapure water for 3 h. Collect the SF solution in the dialysis bag into several 50 mL centrifuge tubes, centrifuge at 8000 rpm / min for 20 min, and centrifuge three times to remove insoluble substances. Then filter through a 0.22 μm filter membrane and store at 4 °C.

[0053] 2. Preparation of silk fibroin microspheres

[0054] (1) Preparation of silk fibroin microsphere core - Ca 2 CO 3 Template

[0055] Prepare 1 mol / L Na 2 CO 3 solution (M = 106, made up to the mark with a 250 mL volumetric flask), 1 mol / L CaCl 2 solution (M = 147, made up to the mark with a 250 mL volumetric flask), add according to the quantitative relationship in the table (mL)

[0056] Solution Name Solution Volume (mL) Water 4 Ethylene Glycol 6 <![CDATA[Sodium 2 Carbon monoxide 3 > 2 <![CDATA[CaCl 2 solution]]> 2

[0057] Add the Na 2 CO 3 solution to the stirred CaCl 2 solution, stir for 1 h without standing.

[0058] (2) Coating with silk fibroin

[0059] ① Centrifuge the prepared silk fibroin solution at 8000 rpm / min for 20 min, and discard the supernatant.

[0060] ② Centrifuge the Ca 2 CO 3 template in step 1 at 3500 rpm / min for 2 min, discard the supernatant, add ethanol - water (ethanol: water volume ratio = 1:1) to about 35 mL, centrifuge at 3500 rpm / min for 2 min, discard the supernatant, and then centrifuge twice with water.

[0061] ③ After discarding the supernatant, add 8 mL of water, resuspend, add 4 mL of silk fibroin, incubate for 1 h (preferably in a vertical suspension instrument), then centrifuge at 3500 rpm / min for 2 min, wash twice with 35 mL of water, each time at 3500 r, 2 min, add 4 mL of water, resuspend, add 20 mL of anhydrous methanol (water: methanol volume ratio = 1:5), vertically suspend, after 30 min, centrifuge at 3500 rpm / min for 2 min, wash twice with 35 mL of water, 3500 rpm / min, 2 min, centrifuge.

[0062] ④ Repeat step ③ for the second - layer coating, then add 50% glutaraldehyde solution to make its concentration 2%, vertically suspend for 1 h, centrifuge at 3500 rpm / min for 2 min, wash twice with water to obtain silk fibroin microspheres, and its electron micrograph is as Figure 4 shown.

[0063] (3) Composite with SDF - 1

[0064] Due to the positive charge of SDF-1 and the negative charge on the surface of silk fibroin microspheres, 2 wt% silk fibroin microspheres and 150 ng / mL SDF-1 were complexed in solution through the principle of positive and negative charge adsorption to form silk fibroin microspheres loaded with SDF-1. After freeze-drying, the microspheres were reserved for use. The Zeta potential diagrams of silk fibroin and silk fibroin microspheres loaded with SDF-1 are as Figure 5 shown, and as Figure 5 shown, the potential of silk fibroin microspheres loaded with SDF-1 increased slightly.

[0065] III. Preparation of injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1 silk fibroin microspheres

[0066] The silk fibroin microspheres loaded with SDF-1, HA-ADH solution containing BMP2 mimetic peptide (HA-CHO-BP solution), and HA-CHO solution containing BMP2 mimetic peptide (i.e., the precursor solution of hyaluronic acid hydrogel containing BMP2 mimetic peptide) were mixed in equal volumes with PBS as the solvent at room temperature to form an injectable hyaluronic acid hydrogel containing BMP2 and SDF-1-silk fibroin microspheres. Its electron micrograph is as Figure 7 shown.

[0067] Example 2

[0068] I. Preparation of precursor solution of hyaluronic acid hydrogel containing BMP2 mimetic peptide

[0069] 1. Preparation of adipic dihydrazide hyaluronic acid (HA-ADH)

[0070] 1. Preparation of adipic dihydrazide-modified hyaluronic acid (HA-ADH)

[0071] 400 mg (90 kDa) of hyaluronic acid (HA) was dissolved in 80 mL of distilled water, and 5.2 g of adipic dihydrazide was added and reacted for 30 min. 0.62 g of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 0.612 g of HOBT (1-hydroxybenzotriazole) were fully dissolved in 80 mL of DMSO (ultrapure water:DMSO = 1:1). After complete dissolution, DMSO was slowly added to the HA solution. The reaction was carried out for 4 h, and the pH was adjusted to 6.8 every 30 min. After the 4-h reaction, the reaction was continued with stirring at room temperature for 24 h. The solution was transferred to a dialysis bag and dialyzed for 3 days, filtered through a 0.22-μm filter membrane, and freeze-dried to obtain solid HA-ADH. 1 The substitution degree of adipic dihydrazide was detected by 1H-NMR, and the calculated value was 30.78%. The 1H-NMR 1 detection of HA and HA-ADH is as Figure 1 , Figure 2 shown.

[0072] 2. Preparation of oxidized hyaluronic acid (HA-CHO)

[0073] Dissolve 400 mg (90 kDa) of hyaluronic acid in 40 mL of distilled water until completely dissolved. Add 206 mg of sodium periodate for oxidation and react for 2 h. Then add an excess of ethylene glycol to terminate the reaction. Transfer the solution to a dialysis bag and dialyze for 3 days. Filter with a 0.22 μm filter membrane and lyophilize to obtain solid HA-CHO.

[0074] Dissolve 50 mg of HA-CHO in 20 mL of 0.25 mol / L hydroxylamine hydrochloride solution. After reacting for 5 h, determine the oxidation degree of HA-CHO by potentiometric titration, and the measured value is 26.32%.

[0075] Use Fourier transform infrared spectroscopy to determine the formation of aldehyde groups in oxidized HA. Take a small amount of lyophilized HA-CHO, HA-ADH and HA, and perform tests by the transmission method. The spectral range of infrared testing is 400 cm -1 -4000 cm -1 , as shown in the infrared spectrum detection Figure 3 as follows

[0076] 3. Preparation of hyaluronic acid hydrogel precursor solution containing BMP2 mimetic peptide

[0077] HA-ADH and HA-CHO are respectively dissolved in a BMP2 mimetic polypeptide solution with a concentration of 4 mg / mL to form 6 wt% solutions, and a HA-ADH solution containing BMP2 mimetic peptide (HA-CHO-BP) and a HA-CHO solution containing BMP2 mimetic peptide (HA-ADH-BP) are prepared. Both are the hyaluronic acid hydrogel precursor solutions containing BMP2 mimetic peptide.

[0078] II. Preparation of silk fibroin microcapsule microspheres loaded with SDF-1

[0079] 1. Purification of silk fibroin

[0080] Weigh 4 g of silk fibroin powder into a 50 mL round-bottom flask, add 20 mL of 9.3 M LiBr solution, place it in a water bath preheated to 60 °C and stir for 4 h. Then transfer it to a 3500 MW dialysis bag and dialyze with ultrapure water for 3 days. Collect the SF solution in the dialysis bag into several 50 mL centrifuge tubes, centrifuge at 8000 rpm / min for 20 min, centrifuge three times to remove insoluble substances, then filter with a 0.22 μm filter membrane and store at 4 °C.

[0081] 2. Preparation of silk fibroin microspheres

[0082] (1) Preparation of silk fibroin microsphere core - Ca 2 CO 3 template

[0083] Prepare 1 mol / L Na 2 CO 3 solution (M = 106, made up to the mark with a 250 mL volumetric flask), 1 mol / L CaCl 2 solution (M = 147, made up to the mark with a 250 mL volumetric flask), and add according to the quantitative relationship in the table (mL)

[0084] Solution Name Solution Volume (mL) Water 4 Ethylene Glycol 6 <![CDATA[Sodium 2 Carbon monoxide 3 > 2 <![CDATA[CaCl 2 solution]]> 2

[0085] Add the Na 2 CO 3 solution to the stirring CaCl 2 solution, stir for 1 h without standing still.

[0086] (2) Coating with silk fibroin

[0087] ① Centrifuge the prepared silk fibroin solution at 8000 rpm / min for 20 min, and discard the supernatant.

[0088] ② Centrifuge the Ca 2 CO 3 template in step 1 at 3500 rpm / min for 2 min, discard the supernatant, add ethanol-water (ethanol: water volume ratio = 1:2) to about 35 mL, centrifuge at 3500 rpm / min for 2 min, discard the supernatant, and then centrifuge twice with water.

[0089] ③ After discarding the supernatant, add 8 mL of water, resuspend, add 4 mL of silk fibroin, incubate for 1 h (preferably in a vertical suspension instrument), then centrifuge at 3500 rpm / min for 2 min, wash twice with 35 mL of water, 3500 r each time for 2 min, add 4 mL of water, resuspend, add 20 mL of anhydrous methanol (water: methanol volume ratio = 1:10), vertically suspend, after 30 min, centrifuge at 3500 rpm / min for 2 min, wash twice with 35 mL of water, 3500 rpm / min for 2 min, and centrifuge.

[0090] ④ Repeat step ③ for the second layer coating, then add 50% glutaraldehyde solution to make its concentration 2%, vertically suspend for 1 h, centrifuge at 3500 rpm / min for 2 min, wash twice with water, and prepare silk fibroin microspheres, the electron micrograph of which is as Figure 4 shown.

[0091] (3) Complexing with SDF-1

[0092] Due to the positive charge of SDF-1 and the negative charge on the surface of silk fibroin microspheres, 2wt% silk fibroin microspheres and 150 ng / mL SDF-1 are combined in solution through the principle of positive and negative charge adsorption to form silk fibroin microspheres complexed with SDF-1. After freeze-drying the microspheres, they are reserved for use. The Zeta potential diagrams of silk fibroin and silk fibroin microspheres complexed with SDF-1 are as shown in Figure 5 shown, and as Figure 5 displayed, the potential of the silk fibroin microspheres complexed with SDF-1 increases slightly.

[0093] III. Preparation of injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1 silk fibroin microspheres

[0094] The silk fibroin microspheres complexed with SDF-1 are mixed with the precursor solution of hyaluronic acid hydrogel containing BMP2 mimetic peptide in equal volume with PBS as the solvent. An injectable hyaluronic acid hydrogel containing BMP2 and SDF-1-silk fibroin can be formed at room temperature, and its electron micrograph is as shown in Figure 7 shown.

[0095] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Preparation method of an injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1, characterized in that, it includes the following steps: 1) Preparation of a hyaluronic acid hydrogel precursor solution containing BMP2 mimetic peptide 1-1) Preparation of adipic dihydrazide modified hyaluronic acid HA-ADH; 1-2) Preparation of oxidized hyaluronic acid HA-CHO; 1-3) Dissolve the above-prepared products HA-ADH and HA-CHO with BMP2 mimetic peptide solution respectively to form an HA-ADH solution containing BMP2 mimetic peptide and an HA-CHO solution containing BMP2 mimetic peptide. These two are the hyaluronic acid hydrogel precursor solutions containing BMP2 mimetic peptide; 2) Preparation of silk fibroin microspheres composite with SDF-1 2-1) Preparation of silk fibroin microspheres: Using CaCO 3 as the core of the silk fibroin microspheres, the silk fibroin is coated on the outside of CaCO 3 ; 2-2) Composite SDF-1 with silk fibroin microspheres in solution through the principle of positive and negative charge adsorption to form silk fibroin microspheres composite with SDF-1; 3) Mix the silk fibroin microspheres composite with SDF-1 with the hyaluronic acid hydrogel precursor solution containing BMP2 mimetic peptide to form an injectable hyaluronic acid hydrogel containing BMP2 mimetic peptide and SDF-1; The specific preparation method of the adipic dihydrazide modified hyaluronic acid HA-ADH is: React hyaluronic acid with adipic dihydrazide under the activation conditions of EDC and HOBT to obtain HA-ADH; The preparation method of the oxidized hyaluronic acid (HA-CHO) is: Oxidize hyaluronic acid with periodic acid to obtain HA-CHO; The specific preparation method of the silk fibroin microspheres is: a. Preparation of silk fibroin microsphere core - CaCO 3 template: Add the Na 2 CO 3 solution to the stirred CaCl 2 solution to form a CaCO 3 template; b. Centrifuge the CaCO prepared in step a, discard the supernatant, add ethanol-water, centrifuge again, discard the supernatant, and then add water and centrifuge multiple times, discarding the supernatant each time; 3 Centrifuge the template, discard the supernatant, add ethanol-water, centrifuge, discard the supernatant, then add water and centrifuge multiple times, discarding the supernatant; c. Add water, resuspend, then add purified silk fibroin, vertically suspend, then centrifuge and wash, add methanol-water, vertically suspend again, centrifuge and wash; d. Repeat step c for multi-layer coating; e. When the ideal layer is wrapped, add glutaraldehyde solution, vertically suspend, centrifuge, and wash with water to obtain silk fibroin microspheres; In step a, CaCO 3 In template preparation, Na 2 CO 3 has a molar ratio of 1:1 with CaCl 2 ; In step b, the volume ratio of ethanol to water added is 1:1 - 1:3; In step c, the volume ratio of methanol to water added is 1:5 - 1:

10.

2. The preparation method according to claim 1, characterized in that, In step c, the specific preparation process of the purified silk fibroin is: Add LiBr solution to silk fibroin powder, place it in a preheated water bath and stir, then transfer it to a dialysis bag and dialyze with ultrapure water, then centrifuge and filter through a membrane for storage.

3. The preparation method according to claim 2, characterized in that, The cut-off molecular weight of the dialysis bag is 3500MW.

4. The preparation method according to claim 2, characterized in that, The pore size of the filter membrane is 0.22μm.

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