CN115845148A

CN115845148A - Thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel and preparation method and application thereof

Info

Publication number: CN115845148A
Application number: CN202211578860.4A
Authority: CN
Inventors: 翁习生; 梁瑞政; 吕泽辉; 王冠云; 卞一鑫; 蔡雪洁
Original assignee: Beijing University of Chemical Technology; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Current assignee: Beijing University of Chemical Technology; Peking Union Medical College Hospital Chinese Academy of Medical Sciences
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2023-03-28

Abstract

The invention discloses a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel as well as a preparation method and application thereof, belonging to the technical field of preparation of medical injectable bone repair materials. The preparation method comprises the steps of 1) synthesizing hydrotalcite nanosheets through coprecipitation; 2) Preparing a hydrotalcite composite material loaded with bone morphogenetic protein 2; 3) Preparing chitosan/silk fibroin/hydrotalcite composite hydrogel; 4) Combining the platelet-derived growth factor with the chitosan/silk fibroin/hydrotalcite composite hydrogel, then blending with a hydrotalcite composite material solution loaded with bone morphogenetic protein 2, and adding a beta-sodium glycerophosphate solution to prepare the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel. The temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material can fix two different growth factors in a layered manner, realize gradual release and promote early formation of blood vessels and continuous regeneration of bone tissues.

Description

Temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel and preparation method and application thereof

Technical Field

The invention relates to the technical field of preparation of medical injectable bone repair materials, in particular to a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel as well as a preparation method and application thereof.

Background

The physical and biochemical coupling relationship between blood vessels and bone cells in bone healing has long been recognized. Therefore, in the field of bone repair, the development of better performing biomaterials, the most important and challenging task is to coordinate the field of bone repair. In angiogenesis, platelet-derived growth factor (PDGF-BB) plays an important role as an important growth factor for angiogenesis, which can target mesenchymal stem cells, promote the generation of new blood vessels, and maintain the stability of new blood vessels. While bone morphogenetic protein 2 (BMP-2) has been widely used as a treatment for orthopedic disorders such as lumbar anterior intervertebral fusion, tibial fractures, etc. due to its excellent osteogenic differentiation properties, it is common to administer a supraphysiological dose of growth factors in order to effectively maintain osteogenic differentiation-inducing levels, both in clinical practice and in basic studies, which may lead to unexpected clinical complications such as ectopic bone formation, formation of cystic tissue, nerve injury, tumor formation, and severe inflammation.

Currently, biomaterial-based strategies have been developed to control sequestration and release of growth factors by non-covalent or covalent growth factor immobilization to reduce their dose and control their local effects. In general, this approach is less common in material preparation because of the negative effects of covalent immobilization on receptor binding and growth factor internalization. The most common way to transport BMP-2 during biomaterial production is by physical adsorption, but it should be noted that physical adsorption of BMP-2 generally has the disadvantages of low loading efficiency, rapid release, molecular aggregation, and degeneration of growth factors. In addition, the growth factors may bind to the biomaterial by non-covalent means such as hydrogen bonding, van der waals forces, electrostatic forces, etc. to delay diffusion of the growth factors. Like most growth factors, BMP-2 (isoelectric point = 9.15) and PDGF-BB (isoelectric point = 9.39) carry a net positive charge. According to previous reports, positively charged growth factors can interact with negatively charged N-/O-sulfate and carboxylate salts of GAG chains, and growth factors can exhibit strong binding affinity to glycosaminoglycans such as hyaluronic acid, heparin, and chondroitin sulfate. For example, chondroitin sulfate (ChS) is a linear and negatively charged polysaccharide whose negatively charged sulfate groups are bound to positively charged growth factors.

The hydrogel is a 3D structural biomaterial capable of retaining a large amount of water, and the unique three-dimensional structure of the hydrogel enables the hydrogel to have wide application in the biomedical field, particularly for the research on drug delivery and some tissue engineering. In contrast to implant scaffolds, injectable hydrogels can be implanted by minimally invasive injection to fill irregular defect shapes in the body without the need for pre-shaping. Among them, some thermoresponsive injectable hydrogels have the unique ability to switch their conformation at physiological temperatures (sol-gel), while ensuring high porosity, which favors angiogenesis, and exchange of signal molecules or growth factors to stimulate tissue repair and regeneration.

Chitosan, a natural polymer, when complexed with glycerophosphate, can form a thermally responsive injectable hydrogel, which is also used in conjunction with other biomaterials (e.g., silk fibroin, hydroxyapatite, etc.) to enhance the complexing properties of the hydrogel, thereby better repairing damaged tissues.

Therefore, how to disclose a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material with layered and fixed growth factors, which avoids clinical complications caused by the administration of a growth factor with a supraphysiological dose, overcomes the defects of low loading efficiency, quick release, molecular aggregation, growth factor denaturation and the like of BMP-2 physical adsorption, and is a difficult problem to be solved in the field.

Disclosure of Invention

The invention aims to provide a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel as well as a preparation method and application thereof, so as to avoid clinical complications caused by administration of a growth factor with an ultraphysiological dose and overcome the defects of low loading efficiency, quick release, molecular aggregation, growth factor denaturation and the like existing in physical adsorption of BMP-2.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel, which comprises the following steps:

1) Mixing a magnesium strontium iron salt solution, a sodium nitrate solution containing formamide and an alkaline solution, and carrying out coprecipitation to synthesize hydrotalcite nanosheets;

2) Mixing the bone morphogenetic protein 2 solution with the chondroitin sulfate solution, combining, adding the hydrotalcite nanosheet solution, blending, combining, and centrifuging to obtain the hydrotalcite composite material loaded with the bone morphogenetic protein 2;

3) In acetic acid water solution, blending chitosan, silk fibroin powder and hydrotalcite nano-sheets at low temperature, and then adding precooled beta-sodium glycerophosphate solution for mixing to prepare chitosan/silk fibroin/hydrotalcite composite hydrogel;

4) Mixing a platelet-derived growth factor solution with chitosan/silk fibroin/hydrotalcite composite hydrogel, combining, blending with a hydrotalcite composite material solution loaded with bone morphogenetic protein 2, adding a beta-sodium glycerophosphate solution, and finally forming temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel;

wherein, the step 2) and the step 3) are not in sequence.

Preferably, the magnesium strontium iron salt solution in the step 1) is prepared by mixing magnesium salt, strontium salt, iron salt and water;

the magnesium salt, the strontium salt and the iron salt are respectively nitrate or chloride of magnesium, strontium and iron;

the total concentration of magnesium salt, strontium salt and iron salt in the magnesium-strontium-iron salt solution is 20-40 mmol/L;

the molar ratio of magnesium ions, strontium ions and iron ions in the magnesium-strontium-iron salt solution is 0.8-3.2;

the sodium nitrate solution containing formamide is a sodium nitrate aqueous solution containing formamide; the concentration of formamide is 4-6 mol/L, and the concentration of sodium nitrate is 9-12 mmol/L;

the alkaline solution contains sodium hydroxide aqueous solution or potassium hydroxide aqueous solution, and the concentration is 0.25-0.35 mmol/mL.

Preferably, in step 1), the mixing manner is as follows: dropwise adding the magnesium strontium iron salt solution and the alkaline solution into the sodium nitrate solution containing formamide;

the dropping speed of the magnesium strontium iron salt solution is 1-3 mu L/s;

the dropping speed of the alkaline solution is 1-3 mu L/s;

the atmosphere of the coprecipitation is protective atmosphere, the pH value of the solution is 9-10, and the temperature is 75-85 ℃.

Preferably, in the step 2), the chondroitin sulfate solution is an aqueous solution of chondroitin sulfate, and the concentration is 0.08-0.12 mg/mL;

the bone morphogenetic protein 2 solution is PBS buffer solution of the bone morphogenetic protein 2, and the concentration of the bone morphogenetic protein 2 solution is 0.08-0.12 mg/mL;

the hydrotalcite nano-sheet solution is an aqueous solution of hydrotalcite nano-sheets, and the concentration is 0.8-1.2 mg/mL;

the volume ratio of the hydrotalcite nanosheet solution to the chondroitin sulfate solution to the bone morphogenetic protein 2 solution is 8-12: 0.5-1.5: 0.05 to 0.15;

preferably, in the step 2), the speed of the centrifugation is 8000-10000 rpm.

Preferably, the concentration of the acetic acid aqueous solution in the step 3) is 0.08-0.12 mol/L;

the low temperature is 0-4 ℃;

in the system finally obtained in the step 3), the mass concentration of chitosan is 10-30 g/L, the mass concentration of silk fibroin is 5-15 g/L, and the mass concentration of hydrotalcite nano-sheets is 0.0125-40 g/L;

the mass concentration of the added precooled beta-sodium glycerophosphate solution is 40-60 g/L; the pre-cooling temperature is-2.5-6.5 ℃;

the volume ratio of the acetic acid water solution to the added precooled sodium beta-glycerophosphate solution is 8-9.

Preferably, in the step 4), the platelet-derived growth factor solution is an aqueous solution of platelet-derived growth factor, and the concentration is 80-120 mug/mL;

the hydrotalcite composite material solution loading the bone morphogenetic protein 2 is an aqueous solution of the hydrotalcite composite material loading the bone morphogenetic protein 2, and the concentration is 10-12 mg/ml;

the concentration of the beta-sodium glycerophosphate solution is 30-70 mg/ml.

Preferably, the volume ratio of the platelet-derived growth factor solution, the chitosan/silk fibroin/hydrotalcite composite hydrogel, the bone morphogenetic protein 2-loaded hydrotalcite composite material solution and the beta-sodium glycerophosphate solution in the step 4) is 0.005-0.015: 0.05 to 0.15.

The invention also aims to provide the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel prepared by the preparation method.

The invention further aims to provide application of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel in bone tissue engineering.

The invention discloses a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel with layered fixed growth factors, which fixes two different growth factors in a layered manner to realize gradual release and promote early formation of blood vessels and continuous regeneration of bone tissues.

Compared with the prior art, the invention has the following beneficial effects:

(1) The temperature-sensitive injectable hydrogel can realize orthopedic minimally invasive surgery and reduce the risk of trauma.

(2) The hydrotalcite can continuously release active metal ions in the hydrogel to promote the regeneration of bone tissues.

(3) The double growth factors are fixed in a layered mode, and the gradual release of different growth factors is achieved.

(4) Simultaneously has excellent osteogenesis and angiogenisis performances.

Therefore, the synthesized thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel is expected to become an orthopedic surgery biomaterial with application prospect.

Drawings

FIG. 1 is TEM (100 nm), TEM (300 nm) and AFM images of hydrotalcite nanosheets (MgSrFe-LDHs) prepared in step 1) of example 1;

FIG. 2 is a potentiometric diagram of MgSrFe-LDHs, chS & BMP-2 and LDH/BMP-2 prepared in step 1), step 2) of example 1 and the BMP-2 loading rate of LDH/BMP-2;

FIG. 3 is a SEM and mapping elemental map of C, CS, CSL/5, CSL/10 prepared in comparative examples 1, 2, 3, 4;

FIG. 4 is a graph of the temperature-sensitive injectable performance of CSL/10 prepared in comparative example 2;

FIG. 5 is a table showing the material compositions of four hydrogels of CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1;

FIG. 6 is a graph showing the ion release profile of the CSL10 hydrogel prepared in comparative example 2 and the release profiles of the growth factors of the CS/B/P-LDH and CS/P-LDH/B hydrogels prepared in comparative example 5 and example 1;

FIG. 7 is an ALP and ARS staining pattern for four hydrogels of CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1;

FIG. 8 is a Calein-AM staining pattern of HUVECs of the four hydrogels of CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1;

FIG. 9 is a surgical diagram of the injection of four hydrogels of CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1 into a skull defect;

FIG. 10 is a plot of Micro-CT, bone volume, and bone mineralization density at 4 and 8 weeks after implantation of the four hydrogels CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5, and example 1 into a skull defect.

Detailed Description

3) In acetic acid water solution, blending chitosan, silk fibroin powder and hydrotalcite nanosheets at low temperature, then adding precooled beta-sodium glycerophosphate solution for mixing, and preparing chitosan/silk fibroin/hydrotalcite composite hydrogel;

wherein, the step 2) and the step 3) are not in sequence.

In the step 1), the magnesium strontium iron salt solution is prepared by mixing magnesium salt, strontium salt, iron salt and water; the water is preferably deionized water;

the magnesium salt, strontium salt and iron salt are preferably nitrate or chloride of magnesium, strontium and iron respectively, and are specifically magnesium nitrate, strontium nitrate, ferric nitrate, magnesium chloride, strontium chloride or ferric chloride;

the total concentration of magnesium salt, strontium salt and iron salt in the magnesium-strontium-iron salt solution is preferably 20-40 mmol/L, more preferably 25-35 mmol/L, and even more preferably 28-32 mmol/L;

the molar ratio of the magnesium ions, the strontium ions and the iron ions in the magnesium strontium iron salt solution is preferably 0.8-3.2: 0.4-0.6;

the sodium nitrate solution containing formamide is a sodium nitrate aqueous solution containing formamide; the concentration of formamide is preferably 4 to 6mol/L, more preferably 4.2 to 5.5mol/L, and even more preferably 4.5 to 5mol/L; the concentration of sodium nitrate is 9 to 12mmol/L, more preferably 9 to 11mmol/L, and still more preferably 10 to 11mmol/L;

the alkaline solution contains an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution, and the concentration is preferably 0.25 to 0.35mmol/mL, more preferably 0.28 to 0.32mmol/mL, and still more preferably 0.29 to 0.31mmol/mL.

In step 1) of the present invention, the mixing is preferably performed in the following manner: dropwise adding the magnesium strontium iron salt solution and the alkaline solution into the formamide-containing sodium nitrate solution;

the dripping speed of the magnesium strontium iron salt solution is preferably 1-3 mu L/s, and more preferably 2 mu L/s;

the dropping speed of the alkaline solution is 1-3 mu L/s, and more preferably 2 mu L/s;

the coprecipitation atmosphere is a protective atmosphere, preferably a nitrogen atmosphere; the pH value of the solution is preferably 9-10, and more preferably 9.5; the temperature is preferably 75 to 85 ℃, more preferably 78 to 82 ℃, and still more preferably 80 ℃.

In the step 1) of the invention, an oil bath is adopted to provide constant temperature for coprecipitation, so as to ensure the stable reaction.

In the step 1), during the synthesis process, the metal salt and the alkali solution generate white floccules, which are hydrotalcite nanosheets.

In the present invention, in step 1), the coprecipitation is preferably performed under magnetic stirring conditions, and the stirring speed is preferably 1000 to 4000rpm, more preferably 2000 to 3000rpm, and even more preferably 2250 to 2750rpm; the stirring time is preferably 20 to 40min, more preferably 25 to 35min, and still more preferably 30min.

In the step 1), after the coprecipitation is finished, preferably cooling the obtained system to room temperature, and sequentially performing centrifugation, washing and dialysis to obtain hydrotalcite nanosheets;

the centrifugation speed is 6500-7500 rpm, more preferably 6800-7200 rpm, and more preferably 7000rpm;

the washing is ethanol washing and ultrapure water washing in sequence, wherein the number of times of ethanol washing is preferably 2-5, more preferably 3-4, and even more preferably 3; the number of times of the ultrapure water washing is preferably 2 to 5, more preferably 3 to 4, and still more preferably 3;

the dialysis bag used for dialysis is preferably 3000 to 5000Da, more preferably 3200 to 4500Da, and still more preferably 3500 to 4000Da.

In step 2) of the present invention, the chondroitin sulfate solution is preferably an aqueous solution of chondroitin sulfate, and the concentration is preferably 0.08 to 0.12mg/mL, more preferably 0.09 to 0.11mg/mL, and even more preferably 0.1mg/mL;

the bone morphogenetic protein 2 solution is preferably a PBS (phosphate buffered saline) solution of the bone morphogenetic protein 2, and the concentration of the bone morphogenetic protein 2 solution is preferably 0.08-0.12 mg/mL, more preferably 0.09-0.11 mg/mL, and more preferably 0.1mg/mL;

the hydrotalcite nanosheet solution is preferably an aqueous solution of hydrotalcite nanosheets, and the concentration is preferably 0.8-1.2 mg/mL, more preferably 0.9-1.1 mg/mL, and even more preferably 1mg/mL;

the volume ratio of the hydrotalcite nanosheet solution to the chondroitin sulfate solution to the bone morphogenetic protein 2 solution is preferably 8-12: 0.5-1.5: 0.05 to 0.15, more preferably 9 to 11:0.8 to 1.2:0.08 to 0.12, more preferably 10:1:0.1.

in step 2) of the present invention, the bone morphogenetic protein 2 solution is preferably mixed with the chondroitin sulfate solution at 3 to 5 ℃, more preferably at 4 ℃.

In the step 2) of the invention, chondroitin sulfate (ChS) and bone morphogenetic protein 2 (BMP-2) are further combined with biological materials, which is a better way to slowly and continuously release BMP-2, so that BMP-2 exists in the whole bone repair process, and the continuous regeneration of bone is promoted.

In step 2) of the present invention, the blending and combining manner is preferably stirring, and the stirring speed is preferably 500 to 800rpm, more preferably 550 to 700rpm, and even more preferably 600 to 650rpm; the stirring time is preferably 20 to 40min, more preferably 25 to 35min, and still more preferably 30min.

In step 2) of the present invention, the rate of the centrifugation is preferably 8000 to 10000rpm, more preferably 8200 to 9500rpm, and still more preferably 8800 to 9200rpm.

In step 3) of the present invention, the low-temperature blending is preferably performed by dissolving chitosan in an aqueous solution of acetic acid under low temperature conditions and keeping stirring until the chitosan is completely dissolved. Slowly adding the silk fibroin powder into the chitosan solution, and stirring to obtain the thermal response hydrogel prepolymer solution. The sonication removed excess bubbles from the solution. Simultaneously, adding hydrotalcite nanosheets and mixing in a vortex mode;

the mode of adding the hydrotalcite nanosheets is preferably an aqueous solution added with the hydrotalcite nanosheets, and the concentration is preferably 8 to 12mg/mL, more preferably 9 to 11mg/mL, and even more preferably 10mg/mL.

The stirring speed until the chitosan is completely dissolved is preferably 3000-6000 rpm, more preferably 4000-5000 rpm, and even more preferably 4250-4750 rpm; the stirring time is preferably 20 to 30 hours, more preferably 22 to 28 hours, and even more preferably 24 to 26 hours;

the stirring speed of the thermal response hydrogel prepolymer solution obtained by stirring is preferably 1000 to 4000rpm, more preferably 2000 to 3000rpm, and even more preferably 2250 to 2750rpm; the stirring time is preferably 1.5 to 3 hours, more preferably 1.8 to 2.5 hours, and even more preferably 2 to 2.2 hours;

the ultrasonic frequency of the ultrasonic treatment is preferably 80-120 Hz, more preferably 90-110 Hz, and more preferably 95-105 Hz;

the rotation speed of the vortex mixing is preferably 4000 to 6000rpm, more preferably 4500 to 5500rpm, and more preferably 4800 to 5200rpm; the time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.8 hours, and even more preferably 1.0 to 1.5 hours;

in step 3) of the present invention, the concentration of the aqueous acetic acid solution is preferably 0.08 to 0.12mol/L, more preferably 0.09 to 0.11mol/L, and still more preferably 0.1mol/L;

the low temperature is preferably 0-4 ℃, more preferably 1-3 ℃, and more preferably 2 ℃;

in the system finally obtained in the step 3), the mass concentration of the chitosan is preferably 10-30 g/L, more preferably 12-28 g/L, and more preferably 15-25 g/L; the mass concentration of the silk fibroin is preferably 5-15 g/L, more preferably 8-12 g/L, and more preferably 10g/L; the mass concentration of the hydrotalcite nanosheets is preferably 0.0125-40 g/L, more preferably 0.05-35 g/L, and even more preferably 0.1-30 g/L.

The mass concentration of the added precooled beta-sodium glycerophosphate solution is preferably 40 to 60g/L, further preferably 45 to 55g/L, and more preferably 48 to 52g/L; the pre-cooling temperature is preferably-2.5-6.5 ℃, more preferably-1-5 ℃, and more preferably 0-4 ℃;

the volume ratio of the aqueous acetic acid solution to the added precooled sodium beta-glycerophosphate solution is preferably 8-9, more preferably 8.3-8.6.

In step 4) of the present invention, the platelet-derived growth factor solution is preferably an aqueous solution of platelet-derived growth factor, and the concentration is preferably 80 to 120. Mu.g/mL, more preferably 90 to 110. Mu.g/mL, and still more preferably 95 to 105. Mu.g/mL;

the hydrotalcite composite material solution loaded with the bone morphogenetic protein 2 is preferably an aqueous solution of the hydrotalcite composite material loaded with the bone morphogenetic protein 2, and the concentration of the hydrotalcite composite material solution is preferably 10-12 mg/ml, more preferably 10-11 mg/ml, and even more preferably 10mg/ml;

the concentration of the sodium beta-glycerophosphate solution is 30 to 70mg/ml, more preferably 35 to 65mg/ml, and still more preferably 40 to 60mg/ml.

In step 4) of the invention, the volume ratio of the platelet-derived growth factor solution, the chitosan/silk fibroin/hydrotalcite composite hydrogel, the hydrotalcite composite material solution loaded with bone morphogenetic protein 2 and the beta-sodium glycerophosphate solution is preferably 0.005-0.015: 0.05 to 0.15; more preferably from 0.008 to 0.014: 0.08 to 0.14; more preferably from 0.01 to 0.012, and from 7.5 to 8.5:0.1 to 0.12.

In step 4) of the present invention, the mixing is preferably performed under magnetic stirring, and the temperature of the magnetic stirring is preferably-2 to 2 ℃, more preferably-1 to 1 ℃, and still more preferably 0 ℃; the stirring speed is preferably 1000 to 4000rpm, more preferably 2000 to 3000rpm, and still more preferably 2250 to 2750rpm; the stirring time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.8 hours, and still more preferably 1.0 to 1.5 hours.

In the step 4) of the present invention, the hydrotalcite composite material solution loaded with bone morphogenetic protein 2 is preferably blended by stirring at a speed of preferably 1000 to 4000rpm, more preferably 2000 to 3000rpm, and even more preferably 2250 to 2750rpm; the stirring time is preferably 0.5 to 2 hours, more preferably 0.8 to 1.8 hours, and still more preferably 1.0 to 1.5 hours.

In the step 4), the reaction condition after adding the beta-sodium glycerophosphate solution is preferably water bath heating; the temperature is preferably 36 to 38 deg.C, more preferably 37 deg.C.

The invention provides application of a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material with growth factors fixed in a layered manner in preparation of injectable bone surgery biomaterials. The method of the present invention is not particularly limited, and the method may be applied according to a method known in the art.

The invention provides a preparation method of a temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material with layered and fixed growth factors and application of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material in bone tissue engineering. The temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material with layered fixed growth factors can realize the gradual release of different growth factors so as to promote the generation of prophase blood vessels and the continuous regeneration of bones; the temperature-sensitive injectable material realizes the possibility of minimally invasive surgery treatment, realizes the gelation of the gel material in a physiological environment after being injected into a body, releases bioactive ions along with the degradation of the material and induces osteogenic differentiation. Therefore, the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel material with the synthesized growth factors fixed in a layered manner is expected to become an orthopedic operation biomaterial with application prospect.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1) Adding 0.16mmol Mg (NO) ₃ ) ₂ ·6H ₂ O、0.04mmolSr(NO ₃ ) ₂ And 0.10 mmoleFe (NO) ₃ ) ₃ Dissolving in 10mL of deionized water to obtain a magnesium strontium iron salt solution;

dissolving 0.1mmol of sodium nitrate and 50mmol of formamide in 10mL of deionized water to obtain a sodium nitrate solution containing formamide;

dissolving 0.0025mol of NaOH in 10mL of deionized water to obtain a sodium hydroxide solution;

N ₂ under the atmosphere, the magnesium strontium iron salt solution and the sodium hydroxide solution are simultaneously dripped into the sodium nitrate solution containing formamide at the dripping speed of 2 mu L/s, stirred in an oil bath at the temperature of 80 ℃ until dripping is finished, and then stirred magnetically for 30min (2000 rpm); and naturally cooling the system to room temperature, collecting the obtained sample by centrifugation (6800 rpm), washing the sample with absolute ethyl alcohol and ultrapure water for three times respectively in sequence, and further dialyzing (3500 Da) to remove formamide to obtain the hydrotalcite nanosheet (MgSrFe-LDHs).

2) Electrostatic adsorption was performed using negatively charged chondroitin sulfate (ChS) to bind MgSrFe-LDHs and BMP-2. First, in order to synthesize ChS & BMP-2 complex, bone morphogenetic protein 2 (BMP-2) was dissolved in 50. Mu.L of sterile PBS buffer to obtain BMP-2 solution (0.1 mg/mL), and then 1mL of aqueous ChS solution (0.1 mg/mL) was added at 4 ℃ and continuously stirred at 600rpm for 0.5 hour to prepare ChS & BMP-2 complex. Then, 10mL of an aqueous solution (1 mg/mL) of the hydrotalcite nanosheets is transferred into a ChS & BMP-2 solution and stirred for 6 hours, and centrifuged at 9000rpm to obtain the bone morphogenetic protein 2-loaded hydrotalcite composite material (LDH/BMP-2 composite).

3) At 0 deg.C, 0.2g of chitosan was first dissolved in 8mL of aqueous acetic acid (0.1 mol/L) and stirring was maintained for 24 hours (3500 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.1 g) powder was slowly added to the chitosan solution, and stirred for another 2 hours (3800 rpm) to obtain a heat-responsive hydrogel prepolymer solution. Sonication (100 Hz) was used to remove bubbles. At the same time, 1mL of an aqueous solution of hydrotalcite nanosheets (MgSrFe-LDHs) (10 mg/mL) was added and vortex mixed (6000 rpm) for 30 minutes. And finally, adding 1mL of beta-sodium glycerophosphate (beta-GP, 50 g/L) solution precooled at 0 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel with the hydrotalcite proportion of 0.1% (wt/vol).

4) Dissolving 10 mu of LPDGF-BB aqueous solution (100 mu g/mL) in 8mL of thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel, magnetically stirring at 0 ℃ for 30 minutes (4000 rpm), adding 1mL of LDH/BMP-2 aqueous solution (10 mg/mL), stirring at 4000rpm for 30 minutes, adding 1mL of beta-GP solution (50 mg/mL), and forming the thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CS/P-LDH/B) with double growth factor layered immobilization under the water bath condition of 37 ℃.

Example 2

1) Adding 0.2mmol Mg (NO) ₃ ) ₂ ·6H ₂ O、0.06mmolSr(NO ₃ ) ₂ And 0.14 mmoleFe (NO) ₃ ) ₃ Dissolving in 10mL of deionized water to obtain a magnesium strontium iron salt solution;

dissolving 0.09mmol of sodium nitrate and 40mmol of formamide in 10mL of deionized water to obtain a sodium nitrate solution containing formamide;

dissolving 0.003mol KOH in 10mL of deionized water to obtain a potassium hydroxide solution;

N ₂ under the atmosphere, the magnesium strontium iron salt solution and the sodium hydroxide solution are simultaneously dripped into the sodium nitrate solution containing formamide at the dripping speed of 1 mu L/s, stirred in an oil bath at the temperature of 75 ℃ until dripping is finished, and then stirred for 40min (1 min) by magnetic force000 rpm); and naturally cooling the system to room temperature, collecting the obtained sample by centrifugation (6500 rpm), then sequentially washing the sample twice with absolute ethyl alcohol and ultrapure water respectively, and further dialyzing (4000 Da) to remove formamide to obtain the hydrotalcite nanosheet (MgSrFe-LDHs).

2) Electrostatic adsorption was performed using negatively charged chondroitin sulfate (ChS) to bind MgSrFe-LDHs and BMP-2. First, in order to synthesize ChS & BMP-2 complex, bone morphogenetic protein 2 (BMP-2) was dissolved in 100. Mu.L of sterile PBS buffer to obtain BMP-2 solution (0.08 mg/mL), and then 1mL of aqueous ChS solution (0.08 mg/mL) was added at 3 ℃ and continuously stirred at 500rpm for 40min to prepare ChS & BMP-2 complex. And secondly, transferring 10mL of aqueous solution (0.8 mg/mL) of the hydrotalcite nanosheets into the ChS & BMP-2 solution, stirring for 7 hours, and centrifuging at 8000rpm to obtain the hydrotalcite composite material (LDH/BMP-2 composite) loaded with the bone morphogenetic protein 2.

3) At 0 deg.C, 0.15g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.08 mol/L) and stirring was maintained for 30 hours (3000 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.08 g) powder was slowly added to the chitosan solution, and stirred for 3 hours (1000 rpm) to obtain a thermo-responsive hydrogel prepolymer solution. Sonication (120 Hz) was used to remove bubbles. Simultaneously, 1mL of an aqueous solution (9 mg/mL) of hydrotalcite nanosheets (MgSrFe-LDHs) was added and vortex mixed (4000) for 2h. And finally, adding 1mL of beta-sodium glycerophosphate (beta-GP, 40 g/L) solution precooled at 1 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel with the hydrotalcite proportion of 0.09% (wt/vol).

4) Dissolving 5 mu of LPDGGF-BB aqueous solution (80 mu g/mL) in 7mL of temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel, magnetically stirring at-2 ℃ for 1.5h (1500 rpm), adding 1mL of LDH/BMP-2 aqueous solution (9 mg/mL) and stirring at 2000rpm for 1h, adding 1mL of beta-GP solution (60 mg/mL), and forming the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CS/P-LDH/B) with double growth factor layered immobilization under the condition of 36 ℃ water bath.

Example 3

1) Adding 0.12mmol Mg (NO) ₃ ) ₂ ·6H ₂ O、0.03mmolSr(NO ₃ ) ₂ And 0.05 mmoleFe (NO) ₃ ) ₃ Dissolving in 10mL of deionized water to obtain a magnesium strontium iron salt solution;

dissolving 0.1mmol of sodium nitrate and 60mmol of formamide in 10mL of deionized water to obtain a sodium nitrate solution containing formamide;

dissolving 0.0035mol of NaOH in 10mL of deionized water to obtain a sodium hydroxide solution;

N ₂ under the atmosphere, the magnesium strontium iron salt solution and the sodium hydroxide solution are simultaneously dripped into the sodium nitrate solution containing formamide at the dripping speed of 3 mu L/s, stirred in an oil bath at the temperature of 85 ℃ until dripping is finished, and then stirred for 20min (4000 rpm) by magnetic force; and then the system is naturally cooled to room temperature, the obtained sample is collected by centrifugation (7400 rpm), and then the sample is washed four times by absolute ethyl alcohol and ultrapure water in sequence, and then further dialyzed (4800 Da) to remove formamide, so as to obtain the hydrotalcite nanosheet (MgSrFe-LDHs).

2) Electrostatic adsorption was performed using negatively charged chondroitin sulfate (ChS) to bind MgSrFe-LDHs and BMP-2. First, in order to synthesize the ChS & BMP-2 complex, bone morphogenetic protein 2 (BMP-2) was dissolved in 50. Mu.L of sterile PBS buffer to obtain a BMP-2 solution (0.1 mg/mL), and then 1mL of an aqueous ChS solution (0.12 mg/mL) was added thereto at 3 ℃ and continuously stirred at 750rpm for 20min to prepare the ChS & BMP-2 complex. And secondly, transferring 10mL of aqueous solution (1 mg/mL) of the hydrotalcite nanosheets into the ChS & BMP-2 solution, stirring for 6 hours, and centrifuging at 10000rpm to obtain the hydrotalcite composite material (LDH/BMP-2 composite) loaded with the bone morphogenetic protein 2.

3) At 0 deg.C, 0.28g of chitosan was first dissolved in 8mL of aqueous acetic acid (0.12 mol/L) and stirring was maintained for 20 hours (5500 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.12 g) powder was slowly added to the chitosan solution, and stirred for another 1.5 hours (2500 rpm) to obtain a thermally responsive hydrogel prepolymer solution. Sonication (85 Hz) was used to remove bubbles. At the same time, 1mL of an aqueous solution of hydrotalcite nanosheets (MgSrFe-LDHs) (12 mg/mL) was added and vortex mixed (5000 rpm) for 1.5h. And finally, adding 1mL of beta-sodium glycerophosphate (beta-GP, 55 g/L) solution precooled at the temperature of 1 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel with the hydrotalcite proportion of 0.12% (wt/vol).

4) Dissolving 10 mu of LPDGF-BB aqueous solution (115 mu g/mL) in 9mL of temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel, magnetically stirring at 1 ℃ for 2h (3000 rpm), adding 1mL of LDH/BMP-2 aqueous solution (12 mg/mL), stirring at 4000rpm for 2h, adding 1mL of beta-GP solution (45 mg/mL), and forming the double-growth-factor layered fixed temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CS/P-LDH/B) under the condition of 37 ℃ water bath.

Comparative example 1

At 0 ℃, 0.2g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.1 mol/L) and kept stirring for 24 hours (3500 rpm) until the chitosan was completely dissolved, to obtain a heat-responsive hydrogel prepolymer solution. And (3) after ultrasonic treatment (100 Hz) is used for removing bubbles, adding 1mL of precooled beta-sodium glycerophosphate (beta-GP, 50 g/L) solution at 0 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan hydrogel (C).

Comparative example 2

At 0 deg.C, 0.2g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.1 mol/L) and stirring (3500 rpm) was maintained for 24 hours until the chitosan was completely dissolved. Then, silk fibroin (0.1 g) powder was slowly added to the chitosan solution, and stirred (3800 rpm) for 2 hours to obtain a heat-responsive hydrogel prepolymer solution. Sonication (100 Hz) was used to remove bubbles. Adding 1mL of beta-sodium glycerophosphate (beta-GP, 50 g/L) solution precooled at 0 ℃, and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin composite hydrogel (CS).

Comparative example 3

At 0 deg.C, 0.2g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.1 mol/L) and stirring was maintained for 24 hours (3500 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.1 g) powder was slowly added to the chitosan solution, and stirred for another 2 hours (3800 rpm) to obtain a heat-responsive hydrogel prepolymer solution. Sonication (100 Hz) was used to remove bubbles. Meanwhile, 1mL of an aqueous solution of hydrotalcite nanosheets (MgSrFe-LDHs) (5 mg/mL) was added and vortex mixed (6000 rpm) for 30 minutes. And finally, adding 1mL of beta-sodium glycerophosphate (beta-GP, 50 g/L) solution precooled at 0 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CSL 5) with the hydrotalcite proportion of 0.05% (wt/vol).

Comparative example 4

At 0 deg.C, 0.2g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.1 mol/L) and stirring was maintained for 24 hours (3500 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.1 g) powder was slowly added to the chitosan solution, and stirred for another 2 hours (3800 rpm) to obtain a heat-responsive hydrogel prepolymer solution. Sonication (100 Hz) was used to remove bubbles. At the same time, 1mL of LDHs (10 mg/mL) was added and vortex mixed (6000 rpm) for 30 minutes. And finally, adding 1mL of 0 ℃ precooled beta-sodium glycerophosphate (beta-GP, 50 g/L) solution and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CSL 10 or CS-LDH) with the hydrotalcite proportion of 0.1% (wt/vol).

Comparative example 5

At 0 deg.C, 0.2g of chitosan was first dissolved in 8mL of an aqueous acetic acid solution (0.1 mol/L) and stirring was maintained for 24 hours (3500 rpm) until the chitosan was completely dissolved. Then, silk fibroin (0.1 g) powder was slowly added to the chitosan solution, and stirred for another 2 hours (3800 rpm) to obtain a heat-responsive hydrogel prepolymer solution. Sonication (100 Hz) was used to remove bubbles. Meanwhile, 1mL of an aqueous solution of hydrotalcite nanosheets (MgSrFe-LDHs) (10 mg/mL) was added and vortex mixed (6000 rpm) for 30 minutes. And finally, adding 1mL of beta-sodium glycerophosphate (beta-GP, 50 g/L) solution precooled at 0 ℃ and uniformly mixing to obtain the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel with the hydrotalcite proportion of 0.1% (wt/vol).

2) Adding 50 mu L of BMP-2 aqueous solution (100 mu g/mL) and 10 mu L of PDGF-BB aqueous solution (100 mu g/mL) into 8mL of temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel, magnetically stirring at 0 ℃ for 30min (4000 rpm), adding 1mL of hydrotalcite (10 mg/mL) and stirring at 4000rpm for 30min, adding 1mL of beta-GP solution (concentration), and forming the dual-growth-factor-doped temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel (CS/B/P-LDH) under the condition of 37 ℃ water bath.

Characterization and testing

1) TEM and AFM tests are performed on the magnesium strontium iron hydrotalcite material prepared in step 1) of example 1, and the results are shown in fig. 1, wherein TEM images can show the hexagonal morphology of the hydrotalcite nanosheets, and the size is about 107nm; the morphology of the hydrotalcite nanosheets is shown by the AFM image, with thicknesses of 4.53, 3.82, and 3.76nm, respectively, as indicated by the height profile labeled in fig. 1 c.

2) The potential potentials of MgSrFe-LDHs, chS & BMP-2 and LDH/BMP-2 prepared in step 1) and step 2) of example 1 and the load rate of BMP-2 were measured, and the results are shown in FIG. 2, and it can be seen from FIG. 2 that MgSrFe-LDHs shows a positive potential (38.4 + -2.9 mV) in an aqueous solution, and after MgSrFe-LDHs loads ChS & BMP-2, the Zeta potential becomes 7.7 + -1.3 mV in water, and similar values were obtained in DMEM medium and PBS buffer as well; the BMP-2 loading efficiency is determined to be over 80% by enzyme-linked immunosorbent assay (ELISA).

3) SEM and element mapping characterization is carried out on C, CS, CSL/5 and CSL/10 prepared by comparative examples 1, 2, 3 and 4, the result is shown in figure 3, the interconnected porous structure of C, CS, CSL5 and CSL10 is confirmed by SEM, and the pore diameter is in the range of 10-150 μm; some waviness and wrinkles appeared on the pore walls of CSL hydrogels (CSL 5 and CSL 10), which would facilitate cell adhesion; the element mapping representatively illustrates that Mg, sr and Fe elements of MgSrFe-LDHs are uniformly distributed in the CSL composite hydrogel.

4) The temperature-sensitive and injectable performance of the CSL/10 prepared in comparative example 2 was tested, and the results are shown in fig. 4. In order to evaluate whether the thermo-responsive CSL hydrogel can maintain the injectability and the temperature-sensitive gel characteristics to adapt to irregular defects in biological tissues, the ex vivo test was performed by injecting the thermo-responsive hydrogel into the defect sites of pork tissues using a 22G needle; after injection using a syringe, the hydrogel solution was observed to quickly and accurately conform to defects in pork tissue without clogging. Even aiming at pork tissues with defects of different shapes, the hydrogel solution can quickly fill the defects after injection and can be successfully cured under the condition of 37 ℃ water bath.

5) The material components of the four hydrogels of CS, CS-LDH, CS/B/P-LDH, and CS/P-LDH/B prepared in comparative examples 2, 4, and 5 and example 1 are explained in detail. As shown in fig. 5, CS group has only chitosan and silk fibroin; the CS-LDH group was supplemented with hydrotalcite (0.1 wt/vol%); the CS/B/P-LDH group directly mixes BMP-2 and PDGF-BB into the hydrogel; the CS/P-LDH/B group is combined with BMP-2 through chondroitin sulfate and then is combined with hydrotalcite to achieve the effect of slowly releasing the BMP-2, and PDGF-BB is directly mixed into hydrogel to realize early release.

6) When the ion release of the CSL10 hydrogel prepared in the comparative example 2 and the growth factor release performance of the CS/B/P-LDH and CS/P-LDH/B hydrogels prepared in the comparative example 5 and the example 1 are tested, as can be seen from FIG. 6, BMP-2 and PDGF-BB both maintain burst release in the CS/B/P-LDH hydrogel, indicating that the growth factors directly embedded in the hydrogel cannot be well preserved; the release rate of PDGF-BB in the CS/P-LDH/B hydrogel is relatively fast, while the release rate of BMP-2 is slow and is sustainable. Specifically, the percentages of release of BMP-2 and PDGF-BB were approximately 15.71% and 64.49%, respectively, within 1 day. The release of BMP-2 becomes stable and sustained over time. By the end of the measurement (35 days), the cumulative release percentage of BMP-2 was about 74.51%.

7) ALP and ARS staining pattern tests on the four hydrogels CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1 were performed to characterize osteogenic properties of the different hydrogel groups, and the results are shown in FIG. 7, ALP is a marker of early osteogenesis, which can increase collagen deposition and mineralization, which are main components of bone matrix at later time points, ALP staining images and quantitative analysis of human mesenchymal stem cells (hBMSCs) on different substrates after 14 days of culture are shown in FIG. 7 a; compared with the CS group alone, the alkaline phosphatase (ALP) activity of hBMSCs on CSL, CS/B/P-LDH and CS/B/P-LDH hydrogel is obviously improved, particularly the ALP activity in the CS/P-LDH/B hydrogel is the highest and is far higher than that of the CSL or even the CS/B/P-LDH group, which indicates that the CS/P-LDH/B can obviously increase the osteogenesis capacity due to the continuous release of BMP-2 and the synergistic action of LDHs; furthermore, the maturity of hBMSCs was determined by characterizing extracellular calcium deposits using alizarin red S staining as a late marker of day 14 osteogenesis; alizarin Red S (ARS) staining pattern showed that mineral nodules produced by hBMSCs in CS/P-LDH/B group were much larger in size and number than the other three groups (fig. 7B), consistent with ALP results, further confirming that sustained release of BMP-2 and synergy of LDHs can promote the osteogenesis of hBMSCs.

8) Calein-AM staining of HUVECs of the four hydrogels of CS, CS-LDH, CS/B/P-LDH, and CS/P-LDH/B prepared in comparative examples 2, 4, and 5 and example 1 was performed to characterize the vascularization ability of different hydrogel groups, and the results are shown in FIG. 8, wherein the HUVECs incubated with the hydrogels of the CS/B/P-LDH and CS/P-LDH/B groups have distinct tubular structures.

9) A skull defect animal model is established, four hydrogels CS, CS-LDH, CS/B/P-LDH and CS/P-LDH/B prepared in comparative examples 2, 4 and 5 and prepared in example 1 are respectively injected into corresponding parts of a skull, as shown in figure 9, and four groups of hydrogels realize rapid gelation when being injected into the skull defect part, so that a hydrogel solid structure is formed.

10 Micro-CT analysis of the

bone regeneration effects

4 and 8 weeks after the implantation of the four hydrogels of CS, CS-LDH, CS/B/P-LDH, CS/P-LDH/B prepared in comparative examples 2, 4, 5 and example 1 into the skull defect showed that the accumulation of new bone in the CS/B/P-LDH and CS/P-LDH/B groups was significantly increased at week 4 as shown in FIG. 10; at week 8, both the CS/B/P-LDH and CS/P-LDH/B hydrogels promoted the formation of new bone from the periphery to the center of the defect area, while the newly formed bone tissue more significantly penetrated to the center of the defect area; quantitative morphological analysis using Micro-CT analysis showed that bone volume/total volume ratio (BV/TV) in CS/P-LDH/B hydrogel was significantly higher than the other three groups (fig. 10B); at the same time, the Bone Mineralization Density (BMD) of the CS/P-LDH/B group was significantly higher than that of the CS/B/P-LDH group (FIG. 10 c), which means that the hydrotalcite composite hydrogel, which allows for the initial release of PDGF-BB and the sustained release of BMP-2, stimulates the accelerated promotion of new bone formation and defect healing.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel is characterized by comprising the following steps:

2) Mixing the bone morphogenetic protein 2 solution with the chondroitin sulfate solution, combining, adding a hydrotalcite nanosheet solution, blending, combining, and centrifuging to obtain a hydrotalcite composite material loaded with the bone morphogenetic protein 2;

wherein, the step 2) and the step 3) are not in sequence.

2. The preparation method of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 1, wherein the magnesium strontium iron salt solution in step 1) is prepared by mixing a magnesium salt, a strontium salt, an iron salt and water;

the magnesium salt, the strontium salt and the ferric salt are respectively nitrate or chloride of magnesium, strontium and iron;

3. The preparation method of the thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 1 or 2, wherein in the step 1), the mixing manner is as follows: dropwise adding the magnesium strontium iron salt solution and the alkaline solution into the formamide-containing sodium nitrate solution;

the dropping speed of the magnesium strontium iron salt solution is 1-3 mu L/s;

the dropping speed of the alkaline solution is 1-3 mu L/s;

4. The preparation method of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 3, wherein in the step 2), the chondroitin sulfate solution is an aqueous solution of chondroitin sulfate, and the concentration is 0.08-0.12 mg/mL;

the volume ratio of the hydrotalcite nanosheet solution to the chondroitin sulfate solution to the bone morphogenetic protein 2 solution is 8-12: 0.5-1.5: 0.05 to 0.15.

5. The preparation method of the thermosensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 1, 2 or 4, wherein in the step 2), the centrifugation rate is 8000-10000 rpm.

6. The preparation method of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 5, wherein the concentration of the aqueous acetic acid solution in the step 3) is 0.08-0.12 mol/L;

the low temperature is 0-4 ℃;

7. The preparation method of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 1, 2, 4 or 6, wherein in the step 4), the platelet-derived growth factor solution is an aqueous solution of platelet-derived growth factor, and the concentration is 80-120 μ g/mL;

the concentration of the beta-sodium glycerophosphate solution is 30-70 mg/ml.

8. The preparation method of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 7, wherein the volume ratio of the platelet-derived growth factor solution, the chitosan/silk fibroin/hydrotalcite composite hydrogel, the hydrotalcite composite material solution loaded with bone morphogenetic protein 2, and the β -sodium glycerophosphate solution in step 4) is 0.005-0.015: 7-9: 0.05 to 0.15.

9. The temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel prepared by the preparation method of any one of claims 1 to 8.

10. The use of the temperature-sensitive injectable chitosan/silk fibroin/hydrotalcite composite hydrogel according to claim 9 in bone tissue engineering.