Regenerated silk fibroin material, and preparation method therefor and use thereof

Abstract

The present invention relates to a silk fibroin material, and a preparation method therefor and a use thereof. The preparation method for the silk fibroin material comprises subjecting a solution containing regenerated silk fibroin to gelation treatment to obtain the silk fibroin material. The present invention has the advantages that the preparation method is simple and efficient, is waste-free and pollution-free throughout the process, exhibits good repeatability, and has the potential value for industrial production.

Description

Regenerated silk fibroin material, preparation method and application TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a regenerated silk fibroin material, a preparation method and application. BACKGROUND

[0002] Silk fibroin is a kind of natural biological protein material, which has good biocompatibility, easy to obtain, good swelling property and natural antibacterial property. Foreign literatures have reported that silk fibroin is used to prepare bone tissue repair materials such as scaffolds, gels and fiber meshes, which have been confirmed to have good biocompatibility in animal experiments. However, due to the slow degradation rate of pure silk fibroin material, and compared with other proteins such as collagen and gelatin, the silk fibroin material lacks bioactive peptide segments, and thus has weak bone induction and bone growth promotion ability.

[0003] Silk fibroin can be used for absorbable bone nails and bone plates, which can be used for internal fixation systems in trauma orthopedics and maxillofacial plastic surgery. Under the background of population aging in China, the number of patients with fractures is increasing, and the demand for bone nails and bone plates will continue to rise, and the development of the industry is highly certain.

[0004] At present, the widely used degradable bone fixation material is mainly high molecular polymer, including polyglycolic acid (PGA), polylactic acid (PLA) and poly-lactic-glycolic acid copolymer (PLGA). For many years, foreign researchers have continuously innovated and researched, and the high molecular degradable bone fixation material has basically met the clinical requirements, but there are still some deficiencies in the PLA type polymer material as an orthopedic internal fixation material. The biggest problem is the postoperative complications of PLA type bone fixation material. The degradation products of PLA type material are glycolic acid and lactic acid, which can participate in the body's sugar metabolism cycle. However, the in vivo degradation process of large size PLA type material (limb bone nails, fixation plates) is easy to form local enrichment of acidic products (glycolic acid and lactic acid), causing immune rejection and bone regeneration problems. It has been reported that the main clinical immune rejection reactions include local swelling, sterile abscess, fistula and bone resorption. In addition, the PLA type material also has the problems of not having bone conduction, insufficient mechanical strength, "passive" overall hydrolysis, "self-catalytic" degradation effect leading to rapid disintegration of the material in the middle and late stages of degradation and large release of acidic products.

[0005] The regenerated silk fibroin material is mainly prepared from cocoon through degumming, dissolution, dialysis and modification operations. Soft materials such as sutures and paste gels made of degummed silk have been widely clinically researched and applied, but there are still many difficulties in the process of hard silk fibroin materials. At present, the main preparation methods of hard silk fibroin materials mainly include solvent (methanol, etc.) recrystallization method, sol-gel method and direct hot forming method.

[0006] But the preparation method also has some problems, resulting in the final scheme is difficult to implement industrialization, for example:

[0007] 1、The current reported based on methanol recrystallization method of silk protein material inside a large number of residual bubble (> 50%), and the mechanical properties and process stability is difficult to guarantee, difficult to prepare large size material, can not be used for product development, seriously restricts the application of silk protein material.

[0008] 2、General methanol recrystallization method after improvement, can solve the problem of bubble and large-scale production, but the process using hexafluoroisopropanol as solvent, not only expensive and harmful to the human body, high removal requirements, inducer methanol in addition to low cost also has the same problem.

[0009] 3、Direct hot pressing forming method can also be prepared into specific shape hard material, but the hot pressing equipment and raw material requirements are too high, large-scale production is difficult.

[0010] 4、The silk protein hard material prepared by the method of natural drying of hydrogel needs to be dried naturally again with other enhancement methods to achieve sufficient mechanical strength, such as UV / riboflavin system, HRP / H2O2 system, chitin / chitosan interpenetrating network, etc. These systems inevitably require more thorough mixing, but silk protein, due to its surfactant-like properties, will generate a large number of bubbles during mixing, especially when preparing large samples for industrialization, bubbles will be more difficult to escape. If the bubbles cannot be fully removed and remain in the gel system, it will seriously affect the performance of the final hard material, especially the mechanical properties.

[0011] Chinese invention patent (CN111956871) discloses a silk protein / gelatin composite material and its use: the silkworm cocoon is immersed in a soap salt solution to degum, dried to prepare degummed silk, and the degummed silk is dissolved in lithium bromide solution, dialyzed to remove salt, and concentrated to prepare a silk protein solution; the silk protein solution is mixed with a gelatin solution, and horseradish peroxidase, hydrogen peroxide solution and glutamine transaminase are added, mixed uniformly to form a glue, and a silk protein / gelatin gel is obtained; the silk protein / gelatin gel is immersed in alcohol and ammonium sulfate solution respectively to obtain a silk protein / gelatin crosslinked gel, which is dried to obtain a columnar or block-shaped embryo.

[0012] In addition, silk fibroin can be applied to the repair of bone defects in the joint, the fixation of artificial joint revision bone transplantation, the reconstruction of ligament in sports trauma, and the transplantation of bone blocks in bone tumor resection. However, the composite material disclosed in the above invention does not have a porous structure, which cannot provide a three-dimensional space for the adhesion, proliferation and migration of bone repair cells, resulting in low efficiency of nutrient and water transport to cells and affecting the osteogenic activity of the material.

[0013] Further, Viviana et al. disclosed a method in J. Actbio. 2018.03.047: degumming of mulberry cocoon by immersing in a soap salt solution, drying to prepare degummed silk, dissolving the degummed silk in a lithium bromide solution, dialysis desalination and concentration to prepare a silk protein solution; adding horseradish peroxidase and hydrogen peroxide solution to the silk protein solution, and transferring into a mold before gelation; slowly adding granular sodium chloride to the solution, and gelating at 37℃; soaking the silk protein gel in purified water, filtering out the undissolved salt particles to obtain a porous gel; removing from the mold, and freeze-drying to obtain the porous gel.

[0014] However, the silk protein scaffold material prepared by the above method does not contain gelatin component, so that the biological activity of the silk protein scaffold material in promoting cell adhesion, differentiation and growth is lower than that of the material containing gelatin component, that is, the stability and biological function of the material are not as good as those of the composite material containing gelatin component.

[0015] At present, there is no effective solution to the problems in the related silk fibroin nail plate system preparation technology, such as the internal residual of a large number of bubbles in the silk protein hard material prepared by the methanol recrystallization method and the natural drying method of hydrogel, the high production cost of the improved methanol recrystallization method of silk, and the difficulty in large-scale production by direct hot pressing forming method, as well as the problems in the bone repair material preparation technology, such as the lack of porous structure in the composite material and the lack of gelatin component in the silk protein scaffold material. SUMMARY

[0016] The purpose of the present application is to solve the problems in the related silk fibroin nail plate system preparation technology, such as the internal residual of a large number of bubbles in the silk protein hard material prepared by the methanol recrystallization method and the natural drying method of hydrogel, the high production cost of the improved methanol recrystallization method of silk, and the difficulty in large-scale production by direct hot pressing forming method, as well as the problems in the bone repair material preparation technology, such as the lack of porous structure in the composite material and the lack of gelatin component in the silk protein scaffold material.

[0017] To achieve the above purpose, the technical scheme adopted by the present application is:

[0018] In a first aspect, the present application provides a preparation method of a regenerated silk fibroin hard material, comprising:

[0019] The solution containing regenerated silk fibroin is subjected to bubble removal treatment to obtain a high-purity silk fibroin solution or a silk fibroin / gelatin composite solution;

[0020] The high-purity silk fibroin solution or the silk fibroin / gelatin composite solution is subjected to gel treatment to obtain a regenerated silk fibroin hard material.

[0021] In some embodiments, the method further comprises:

[0022] The regenerated silk fibroin solution is subjected to bubble removal treatment to remove bubbles inside the regenerated silk fibroin solution, and a high-purity silk fibroin solution is obtained.

[0023] The high-purity silk fibroin solution is subjected to gel treatment to obtain a regenerated silk fibroin hard material.

[0024] In some embodiments, the method further comprises:

[0025] The regenerated silk fibroin solution is mixed with a gelatin solution to obtain a regenerated silk fibroin composite solution;

[0026] The regenerated silk fibroin composite solution is subjected to bubble removal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gelatin composite solution is obtained.

[0027] The regenerated silk fibroin / gelatin composite solution is subjected to gel treatment to obtain a regenerated silk fibroin hard material.

[0028] In some embodiments, the method for preparing the regenerated silk fibroin solution comprises:

[0029] The cocoon is cut into pieces and boiled in a sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained.

[0030] The silk is repeatedly washed with deionized water and dried to obtain silk fibers.

[0031] The silk fibers are dissolved in a lithium bromide solution and filtered to obtain a silk fibroin stock solution.

[0032] The silk fibroin stock solution is subjected to dialysis and desalination to obtain a silk fibroin solution.

[0033] The silk fibroin solution is subjected to reverse dialysis and concentration using polyethylene glycol to obtain a regenerated silk fibroin solution.

[0034] In some embodiments, mixing the regenerated silk fibroin solution with the gelatin solution to obtain a regenerated silk fibroin composite solution comprises:

[0035] Horseradish peroxidase and an aqueous hydrogen peroxide solution are added to the regenerated silk fibroin solution, respectively, to obtain a regenerated silk fibroin composite solution.

[0036] The regenerated silk fibroin composite solution is subjected to mechanical shaking to uniformly mix the regenerated silk fibroin composite solution.

[0037] In some embodiments, mixing the regenerated silk fibroin solution with the gelatin solution to obtain a regenerated silk fibroin composite solution comprises:

[0038] The regenerated silk fibroin solution is mixed with the gelatin solution, and then the hydrogen peroxide aqueous solution, the transglutaminase, and the horseradish peroxidase are sequentially added to obtain a silk fibroin composite solution.

[0039] The regenerated silk fibroin composite solution is mechanically shaken to mix the regenerated silk fibroin composite solution uniformly.

[0040] In some embodiments, the regenerated silk fibroin composite solution is subjected to a bubble removal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0041] The regenerated silk fibroin composite solution is placed in a centrifuge tube.

[0042] The centrifuge tube is placed in a centrifuge for centrifugal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0043] In some embodiments, the regenerated silk fibroin composite solution is subjected to a bubble removal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0044] The regenerated silk fibroin composite solution is horizontally flowed into a mold.

[0045] The mold is placed in an ultrasonic machine for ultrasonic treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0046] In some embodiments, the regenerated silk fibroin composite solution is subjected to a bubble removal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0047] The regenerated silk fibroin composite solution is horizontally flowed into a mold.

[0048] A weight is placed on a panel of the mold to extrude the regenerated silk fibroin composite solution in the mold to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0049] In some embodiments, the regenerated silk fibroin composite solution is subjected to a bubble removal treatment to remove bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0050] The regenerated silk fibroin composite solution is directly gelled in an extrusion mold.

[0051] The mold is placed in a vacuum environment to remove air bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0052] In some embodiments, the regenerated silk fibroin composite solution is treated to remove air bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained, including:

[0053] The regenerated silk fibroin composite solution is horizontally flowed into the mold;

[0054] The mold is placed in a low-temperature environment to freeze the regenerated silk fibroin composite solution, and a composite solution frozen block is obtained;

[0055] The composite solution frozen block is dissolved, and a regenerated silk fibroin composite solution is obtained;

[0056] The above steps are repeated 1-2 times to remove air bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained.

[0057] In some embodiments, the regenerated silk fibroin / gel composite solution is subjected to gel treatment to obtain a regenerated silk fibroin hard material, including:

[0058] The regenerated silk fibroin / gel composite solution is subjected to water bath gel to obtain a gel block;

[0059] The gel block is soaked in a methanol solution or an ethanol solution to induce crystallization of the gel block, and a regenerated silk fibroin hard material is obtained;

[0060] The regenerated silk fibroin hard material is taken out of the methanol solution or the ethanol solution and subjected to freeze-drying.

[0061] In a second aspect, the present application provides a regenerated silk fibroin-containing hard material, including a regenerated silk fibroin hard material and a regenerated silk fibroin / gel hard material.

[0062] The hard material is prepared by the preparation method of the first aspect.

[0063] In a third aspect, the present application provides an internal fixation system, including a bone screw and / or a bone plate.

[0064] The bone screw and / or the bone plate are prepared from the hard material of the second aspect;

[0065] The bone screw and / or the bone plate are processed by the following method: the hard material is shaped into a columnar or block-shaped parent embryo, and is processed by a lathe to obtain the bone screw and / or the bone plate.

[0066] The structural strength of the bone nail and / or bone plate is 15-150 MPa in a dry state and 5-50 MPa in a wet state.

[0067] In a fourth aspect, the present application provides a use of a hard material containing regenerated silk fibroin in the preparation of an internal fixation system, which is applied to trauma orthopedics, plastic surgery (craniomaxillofacial), and the hard material is prepared by the preparation method of the first aspect or the hard material of the second aspect or the internal fixation system of the third aspect.

[0068] In a fifth aspect, the present application provides a preparation method of a composite bone repair material, comprising:

[0069] The cocoon is immersed in a soap salt solution for degumming, and dried to obtain degummed silk, and the degummed silk is dissolved in a lithium bromide solution, dialyzed to remove salt, and concentrated to obtain a regenerated silk fibroin solution;

[0070] The regenerated silk fibroin solution is mixed with a gelatin solution, and an aqueous hydrogen peroxide solution, a transglutaminase, and a horseradish peroxidase are sequentially added to obtain a gel solution;

[0071] The gel solution is mechanically shaken to mix the gel solution uniformly and form uniformly distributed bubbles in the gel solution, and the uniformly mixed gelatin solution is horizontally flowed into a mold to gel, and a silk fibroin / gelatin gel block is obtained;

[0072] The silk fibroin / gelatin gel block is soaked and washed in an ammonium sulfate solution to obtain a silk fibroin / gelatin crosslinked gel;

[0073] The silk fibroin / gelatin crosslinked gel is freeze-dried to obtain a silk fibroin / gelatin composite material.

[0074] In some embodiments, the cocoon is immersed in a soap salt solution for degumming, and dried to obtain degummed silk, and the degummed silk is dissolved in a lithium bromide solution, dialyzed to remove salt, and concentrated to obtain a regenerated silk fibroin solution, which comprises:

[0075] The cocoon is cut into pieces and boiled in a sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained;

[0076] The silk is repeatedly washed with deionized water and dried to obtain silk fibers;

[0077] The silk fibers are dissolved in a lithium bromide solution, and filtered to obtain a silk fibroin stock solution;

[0078] The silk fibroin stock solution is dialyzed and desalted to obtain a silk fibroin solution;

[0079] The silk fibroin solution is reverse-dialyzed and concentrated using polyethylene glycol to obtain a regenerated silk fibroin solution.

[0080] In some embodiments, the gel solution is mechanically shaken so that the gel solution is mixed uniformly and uniform bubbles are formed in the gel solution, the uniformly mixed gelatin solution is advection to gel in the mold, and the silk fibroin / gelatin gel block is obtained.

[0081] The gel solution is mechanically shaken so that the gel solution is mixed uniformly and uniform bubbles are formed in the gel solution, and the uniformly mixed gelatin solution is advection to gel in the mold.

[0082] After the preliminary gelation, the gel is cut and a plurality of silk fibroin / gelatin gel blocks are obtained.

[0083] In some embodiments, the method comprises:

[0084] The cocoon is cut into pieces, boiled in a 0.02M sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained.

[0085] The silk is repeatedly washed with deionized water and dried at 40°C for 24h to obtain silk fibers.

[0086] The silk fibers are dissolved in a 9M lithium bromide solution and filtered to obtain a silk fibroin stock solution.

[0087] The silk fibroin stock solution is dialyzed and desalted under the condition of 10000Da to obtain a silk fibroin solution.

[0088] The silk fibroin solution is reverse dialyzed and concentrated using polyethylene glycol with a concentration of 15% and a molecular weight of 20000Da, and a 10% regenerated silk fibroin solution is obtained.

[0089] The 10% regenerated silk fibroin solution is mixed with a 30% gelatin solution, and 0.5% hydrogen peroxide solution with a dosage of 700μl, 100μl transglutaminase with an enzyme activity of 50U / μl, 700μl horseradish peroxidase with an enzyme activity of 500U / mL are sequentially added to obtain a gel solution.

[0090] The gel solution is mechanically shaken so that the gel solution is mixed uniformly and uniform bubbles are formed in the gel solution, and the uniformly mixed gelatin solution is advection to gel in the mold.

[0091] After the preliminary gelation, the gel is cut and a plurality of silk fibroin / gelatin gel blocks are obtained.

[0092] The silk fibroin / gelatin gel block is immersed and washed in a 15% ammonium sulfate solution to obtain a silk fibroin / gelatin crosslinked gel.

[0093] The silk fibroin / gelatin crosslinked gel is freeze-dried for 5 days to obtain a silk fibroin / gelatin composite material.

[0094] In a sixth aspect, the present application further provides a composite bone repair material prepared by the preparation method of the fifth aspect.

[0095] In some embodiments, the composite bone repair material is in the shape of regular particles, irregular particles, spherical solids, cylindrical solids, cubic solids, rectangular solids, or irregularly shaped solids.

[0096] The composite bone repair material has a porous structure at the micron or nanometer scale, and the porosity of the composite bone repair material is 20% to 90%.

[0097] The structural strength (compressive strength) of the composite bone repair material is 0.1 to 35 MPa.

[0098] In a seventh aspect, the present application further provides a composite bone repair material for preparing a bone filling material, which is applied to the filling and regenerative repair of bone defects in orthopedics, oral surgery, plastic surgery, and neurosurgery cranium.

[0099] The present application has the following technical effects compared with the prior art by adopting the above technical scheme:

[0100] 1. The raw materials silk fibroin and gelatin used in the present application have abundant yield, low price, and are certified medical product raw materials by the US Food and Drug Administration, and have good biocompatibility, biological function, and biodegradability.

[0101] 2. The regenerated silk fibroin solution prepared by the fine degumming, washing, dissolving, dialysis, and concentration steps, and the treatment of removing gas bubbles by centrifugation has extremely high purity; high-purity silk fibroin materials are particularly important in biomedical, tissue engineering, and advanced material applications, because they can reduce the immune response or adverse effects caused by impurities.

[0102] 3. After gelation and induced crystallization treatment, the silk fibroin hard material exhibits excellent mechanical properties, such as higher strength and toughness; these improved properties are due to the stable cross-linking and ordered crystalline structure formed between silk fibroin molecules, which can withstand greater stress and deformation without breaking.

[0103] 4. Silk fibroin is a natural polymer material with excellent biocompatibility. The use of toxic solvents and additives is avoided during the preparation process, further ensuring the biological safety of the material; this makes the silk fibroin hard material have wide application prospects in the field of biomedical medicine, such as drug carriers, tissue repair, and regenerative medicine.

[0104] 5、By adjusting the parameters in the preparation process, such as water bath temperature, time, immersion solvent type and time, the structure and performance of silk fibroin materials can be precisely controlled; this processability enables the material to meet the specific needs of different application scenarios.

[0105] 6、The solvents and materials used in the entire preparation process are mostly recyclable or biodegradable, having little impact on the environment; in addition, the preparation process is relatively simple and cost-controllable, which is conducive to realizing large-scale industrial production.

[0106] 7、Silk fibroin materials not only have excellent physical and chemical properties, but also can be further chemically modified or complex modified to endow them with more functional properties, such as antibacterial, conductive, photosensitive, etc., thereby broadening their application range.

[0107] 8、The horseradish peroxidase, hydrogen peroxide, and glutamine transaminase used in the present application have been realized commercialized mass production. Among them, the glutamine enzyme is obtained from microorganisms, which has more uniform stability and abundant yield than the original mammalian source. Low concentration of horseradish peroxidase and hydrogen peroxide have no toxicity and good biocompatibility. Experiments and literature have proved that the silk protein and gelatin hydrogel crosslinked by horseradish peroxidase and glutamine transaminase have no cytotoxicity and animal toxicity.

[0108] 9、The present application applies horseradish peroxidase / hydrogen peroxide to induce crosslinking between silk protein itself and silk protein-gelatin, applies glutamine transaminase to modify the gelatin side branches, and further induces the formation of small and uniform silk protein beta fold in the obtained hydrogel by sequentially immersing it in 70% alcohol solution and 15% ammonium sulfate solution, and strengthens the modification of gelatin branches, so that the composite material has high strength and compression resistance, and also obtains stability in vivo and in animals.

[0109] 10、On the basis of the existing silk protein material, the present application has the biological function of improving cell adhesion, growth and differentiation by adding gelatin.

[0110] 11、On the basis of the existing silk protein / gelatin material, the present application prepares a porous material, which has the advantages of high porosity and greatly promotes cell growth, such as cell proliferation and migration, and tissue growth, which is very beneficial to the clinical application of tissue engineering technology.

[0111] 12、The composite material prepared by the present application has a certain porosity, which can provide a three-dimensional space for the adhesion, proliferation and migration of bone repair cells, and nutrients and water can also be transported to the cells through the pores; and by compounding gelatin and silk protein to prepare the bone filling material, the osteogenic activity of the material can be enhanced.

[0112] 13. The preparation method is simple and efficient, waste-free and pollution-free, has good repeatability, and has potential value for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0113] FIG. 1 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (I);

[0114] FIG. 2 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (II);

[0115] FIG. 3 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (III);

[0116] FIG. 4 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (IV);

[0117] FIG. 5 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (V);

[0118] FIG. 6 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (VI);

[0119] FIG. 7 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (VII);

[0120] FIG. 8 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (VIII);

[0121] FIG. 9 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (IX);

[0122] FIG. 10 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (X);

[0123] FIG. 11 is a flowchart of a preparation method of regenerated silk fibroin hard material according to an embodiment of the present application (XI);

[0124] FIG. 12 is a schematic diagram of a sample prepared by the preparation method of regenerated silk fibroin hard material of FIGS. 1-11 (I);

[0125] FIG. 13 is a schematic diagram of a sample prepared by the preparation method of regenerated silk fibroin hard material of FIGS. 1-11 (II);

[0126] FIG. 14 is a schematic diagram of the test results of FIGS. 12 and 13 according to the test method for 4.6 mechanical strength in the Chinese national industry recommended standard YY / T 1558.3;

[0127] Fig. 15 is a flow chart of a preparation method of the composite bone repair material according to an embodiment of the present application (I);

[0128] Fig. 16 is a flow chart of a preparation method of the composite bone repair material according to an embodiment of the present application (II);

[0129] Fig. 17 is a flow chart of a preparation method of the composite bone repair material according to an embodiment of the present application (III).

[0130] Fig. 18 is a transmission electron microscope (SEM) morphology diagram of the composite bone repair material according to an embodiment of the present application;

[0131] Fig. 19 is a transmission electron microscope (SEM) diagram of cell adhesion of the composite bone repair material according to an embodiment of the present application;

[0132] Fig. 20 is a column chart of in-vitro pull-out force mechanical test of the silk fibroin / gelatin composite bone nail according to an embodiment of the present application;

[0133] Fig. 21 is a column chart of in-vitro shear force mechanical test of the silk fibroin / gelatin composite bone nail according to an embodiment of the present application. DETAILED DESCRIPTION

[0134] The raw materials of the silk fibroin / gelatin composite material provided by the present application are all natural biological materials. The silk fibroin as a natural biological protein material has good biocompatibility, is easy to obtain, has good swelling property and natural antibacterial property and the like. However, due to the slow degradation rate of pure silk protein material, and compared with other proteins such as collagen, gelatin and the like, the silk protein lacks bioactive peptide segments, and the biological function promotion to cells is weak. Pure gelatin is prone to thermal denaturation and water absorption and swelling. The present application utilizes the characteristics that the silk fibroin is rich in tyrosine, and the gelatin is rich in glutamine and lysine, applies horseradish peroxidase and hydrogen peroxide to oxidize the tyrosine in the silk fibroin to form a double tyrosine crosslinking, applies microbial glutamine transaminase to form glutamine-lysine isopeptide bonds in the gelatin through acyl transfer reaction, and adds alcohol solvent to induce the silk fibroin to form a beta fold and ammonium sulfate solution to strengthen the gelatin branch chain modification. The multiple strategies jointly strengthen the stability and mechanical properties of the silk fibroin / gelatin composite material and endow the material with good biological functions.

[0135] The silk fibroin / gelatin composite bone repair material provided by the present application is a new medical biological polymer material, and various specifications of porous bone repair materials can be prepared through mold adjustment and different freeze-drying processes. In clinical surgery, the bone repair material can not only directly fill the bone defect site, but also can be used in cooperation with the nail plate system to improve the bone healing ability, and the material can be completely biodegraded without secondary surgery.

[0136] In order to evaluate the performance of the silk fibroin / gelatin composite bone repair material, the compression strength of the material is tested in the examples, and the results show that the material can well meet the use requirements of defect filling. In addition, the cell adhesion test verifies that the silk fibroin / gelatin composite bone repair material has good cell compatibility, is beneficial to the adhesion and growth of osteoblasts, and is beneficial to bone healing.

[0137] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. Based on the examples in the application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the application.

[0138] In the examples of the application, if no specific description is made for the temperature of experimental operation, the temperature generally refers to room temperature.

[0139] In this paper, the addition amount, content and concentration of various substances are mentioned, and the percentage content mentioned herein refers to the weight percentage content unless otherwise specified.

[0140] The "solution" mentioned herein refers to an aqueous solution unless otherwise specified.

[0141] Example 1

[0142] In one illustrative embodiment of the application, as shown in Figure 1, a preparation method of silk fibroin hard material is applied to high-purity silk fibroin material, which comprises:

[0143] Step S110, the regenerated silk fibroin solution is subjected to bubble removal treatment to remove the bubbles inside the regenerated silk fibroin solution, and a high-purity silk fibroin solution is obtained;

[0144] Step S120, the high-purity silk fibroin solution is subjected to gel treatment to obtain a silk fibroin hard material.

[0145] As shown in Figure 2, the preparation method of the regenerated silk fibroin solution in step S110 comprises:

[0146] Step S210, the cocoon is cut into pieces and put into a sodium carbonate-sodium bicarbonate buffer solution for boiling degumming treatment, and silk is obtained;

[0147] Step S220, the silk is repeatedly washed with deionized water and dried to obtain silk fibers;

[0148] Step S230, the silk fibers are put into a lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0149] Step S240, the silk fibroin stock solution is subjected to dialysis and desalination to obtain a silk fibroin solution;

[0150] Step S250, the silk fibroin solution is reverse-dialyzed and concentrated by polyethylene glycol to obtain a regenerated silk fibroin solution.

[0151] It should be noted that the concentration of the sodium carbonate-sodium bicarbonate buffer in step S210 is 0.01M-0.05M. Preferably, the concentration of the sodium carbonate-sodium bicarbonate buffer is 0.02M.

[0152] It should be noted that the drying temperature in step S220 is 35-45℃, and the drying time is 20-30h. Preferably, the drying temperature is 340℃, and the drying time is 24h.

[0153] It should be noted that the concentration of the lithium bromide solution in step S230 is 8-10M. Preferably, the concentration of the lithium bromide solution is 9M.

[0154] It should be noted that the dialysis molecular weight in step S240 is 8000-14000Da. Preferably, the dialysis molecular weight is 10000Da.

[0155] It should be noted that the molecular weight of polyethylene glycol in step S250 is 10000-30000Da, and the concentration of polyethylene glycol is 10%-20%. Preferably, the molecular weight of polyethylene glycol is 20000Da, and the concentration of polyethylene glycol is 15%.

[0156] It should be noted that the concentration of the regenerated silk fibroin solution in step S250 is 5%-20%. Preferably, the concentration of the regenerated silk fibroin solution is 10%.

[0157] It should be noted that the silk fibroin stock solution is replaced with 4-6 times of aqueous solution during dialysis in step S250. Preferably, the silk fibroin stock solution is replaced with 5 times of aqueous solution during dialysis.

[0158] As shown in FIG. 3, step S110 includes:

[0159] Step S310, placing the regenerated silk fibroin solution in a centrifuge tube;

[0160] Step S320, placing the centrifuge tube in a centrifuge for centrifugal treatment to remove air bubbles inside the regenerated silk fibroin solution and obtain a high-purity silk fibroin solution.

[0161] It should be noted that the centrifugal speed for treating the silk fibroin composite solution in step S320 is 6000-10000rpm. Preferably, the centrifugal speed for treating the silk fibroin composite solution is 8000rpm.

[0162] It should be noted that the centrifugation time of the silk fibroin composite solution in step S320 is 5-60 min. Preferably, the centrifugation time of the silk fibroin composite solution is 30 min.

[0163] It should be noted that during the centrifugation of the regenerated silk fibroin solution, centrifugation and gel treatment can be carried out simultaneously, i.e. gel blocks can be obtained after centrifugation.

[0164] As shown in FIG. 4, further, step S110 further comprises:

[0165] Step S410, the regenerated silk fibroin solution is horizontally flowed into the mold;

[0166] Step S420, the mold is placed in an ultrasonic machine for ultrasonic treatment to remove the bubbles inside the regenerated silk fibroin solution, and a high-purity silk fibroin solution is obtained.

[0167] It should be noted that the ultrasonic frequency of the high-purity silk fibroin solution in step S420 is 30-100 Hz. Preferably, the ultrasonic frequency of the high-purity silk fibroin solution is 70 Hz.

[0168] As shown in FIG. 5, further, step S110 further comprises:

[0169] Step S510, the regenerated silk fibroin solution is horizontally flowed into the mold;

[0170] Step S520, a weight is placed on the panel of the mold to extrude the regenerated silk fibroin solution in the mold to remove the bubbles inside the regenerated silk fibroin solution, and a high-purity silk fibroin solution is obtained.

[0171] It should be noted that the extrusion time of the high-purity silk fibroin solution in step S520 is 30-180 min. Preferably, the extrusion time of the high-purity silk fibroin solution is 120 min.

[0172] As shown in FIG. 6, further, step S110 further comprises:

[0173] Step S610, the regenerated silk fibroin solution is horizontally flowed into the mold;

[0174] Step S620, the mold is placed in a vacuum environment to remove the bubbles inside the regenerated silk fibroin solution, and a high-purity silk fibroin solution is obtained.

[0175] It should be noted that the vacuum degree of the high-purity silk fibroin solution in step S620 is 0.1-0.3 MPa, and the vacuum time is 30-180 min. Preferably, the vacuum degree of the high-purity silk fibroin solution is 0.2 MPa, and the vacuum time is 120 min.

[0176] Further, as shown in FIG. 7, step S110 further comprises:

[0177] Step S710, horizontally flow the regenerated silk fibroin solution into the mold;

[0178] Step S720, place the mold in a low-temperature environment, freeze the regenerated silk fibroin solution, and obtain a solution block;

[0179] Step S730, dissolve the solution block, and obtain a regenerated silk fibroin solution;

[0180] Repeat the above steps 1-2 times to remove the bubbles inside the regenerated silk fibroin solution, and obtain a high-purity silk fibroin gel.

[0181] It should be noted that the freezing temperature for processing the high-purity silk fibroin solution in step S720 is -50 to -80°C, and the freezing time is 3-7 days. Preferably, the freezing temperature for processing the high-purity silk fibroin solution is -65°C, and the freezing time is 5 days.

[0182] It should be noted that steps S710-S730 are repeated twice to remove the bubbles inside the regenerated silk fibroin solution, and obtain a high-purity silk fibroin solution.

[0183] It should be noted that the method for removing bubbles in step S110 can also be processed in a combined manner, for example:

[0184] 1. The centrifugal processing method (steps S310-S320), the ultrasonic processing method (steps S410-S420), the constant pressure processing method (steps S510-S520), the vacuum processing method (steps S610-S620), and the repeated freeze-thaw processing method (steps S710-S730) can be combined with each other in pairs (such as ultrasonic processing + vacuum processing, extrusion processing + vacuum processing, repeated freeze-thaw processing + centrifugal processing, etc.).

[0185] 2. The centrifugal processing method, the ultrasonic processing method, the constant pressure processing method, the vacuum processing method, and the repeated freeze-thaw processing method can be combined with any three (such as repeated freeze-thaw processing + ultrasonic processing + vacuum processing, repeated freeze-thaw processing + extrusion processing + vacuum processing, etc.).

[0186] As shown in FIG. 8, step S120 comprises:

[0187] Step S810, water bath gel the high-purity silk fibroin solution, and obtain a gel block;

[0188] Step S820, soaking the gel block in a methanol solution or an ethanol solution to induce crystallization of the gel block, and obtaining a silk fibroin hard gel block;

[0189] Step S830, taking the regenerated silk fibroin hard gel block out of the methanol solution or the ethanol solution, and drying the regenerated silk fibroin hard gel block.

[0190] It should be noted that the water bath temperature in step S410 is 30-40℃, and the water bath time is 6-24h. Preferably, the water bath temperature is 37℃, and the water bath time is 12h.

[0191] It should be noted that the soaking time in step S420 is 24-36h. Preferably, the soaking time is 30h.

[0192] It should be noted that the drying time in step S430 is 4-7 days. Preferably, the drying time is 5 days.

[0193] The present application has the following advantages:

[0194] 1. The raw material silk fibroin used in the present application is abundant in production, low in price and certified by the US Food and Drug Administration as a medical product raw material, and has good biocompatibility, biological function and biodegradability.

[0195] 2. The silk fibroin solution prepared by the steps of fine degumming, washing, dissolving, dialysis and concentration, and the treatment of removing air bubbles by centrifugation, has very high purity; high-purity silk fibroin material is particularly important in biomedical, tissue engineering and advanced material applications, because it can reduce the immune response or adverse effects caused by impurities.

[0196] 3. After gelation and induced crystallization treatment, the silk fibroin hard material exhibits excellent mechanical properties, such as high strength and toughness; these properties are improved due to the stable cross-linking and ordered crystalline structure formed between silk fibroin molecules, which can withstand greater stress and deformation without breaking.

[0197] 4. As a natural polymer material, silk fibroin has excellent biocompatibility. The use of toxic solvents and additives is avoided during the preparation process, further ensuring the biological safety of the material; this makes the silk fibroin hard material have wide application prospects in the field of biomedical medicine, such as drug carriers, tissue repair and regenerative medicine.

[0198] 5. By adjusting the parameters in the preparation process, such as water bath temperature, time, soaking solvent type and time, the structure and properties of the silk fibroin hard material can be precisely controlled; this processability makes the material meet the specific needs of different application scenarios.

[0199] 6. Most of the solvents and materials used in the entire preparation process are recyclable or biodegradable, resulting in minimal environmental impact. In addition, the preparation process is relatively simple and the cost is controllable, which is conducive to achieving large-scale industrial production.

[0200] 7. Silk fibroin rigid materials not only have excellent physical and chemical properties, but can also be further chemically modified or composite modified to give them more functional properties, such as antibacterial, conductive, and photosensitive properties, thereby broadening their application range.

[0201] Example 2

[0202] This embodiment is a modified embodiment of Embodiment 1. The main difference between this embodiment and Embodiment 1 is that it further includes mixing the regenerated silk fibroin solution with the gelatin solution to obtain a regenerated silk fibroin / gelatin composite solution.

[0203] As shown in Figure 9, a method for preparing a rigid silk fibroin material, applied to silk fibroin / gelatin composite materials, includes:

[0204] Step S910: Mix the regenerated silk fibroin solution with the gelatin solution to obtain a regenerated silk fibroin / gelatin composite solution;

[0205] Step S920: The regenerated silk fibroin / gelatin composite solution is subjected to bubble removal treatment to remove the bubbles inside the regenerated silk fibroin / gelatin composite solution and obtain the regenerated silk fibroin / gelatin gel composite solution.

[0206] Step S930: The regenerated silk fibroin / gelatin gel composite solution is subjected to gel treatment to obtain regenerated silk fibroin / gelatin gel block.

[0207] Step S940: Remove and dry the regenerated silk fibroin / gelatin gel block to obtain a regenerated silk fibroin / gelatin rigid material.

[0208] It should be noted that the preparation method of the regenerated silk fibroin solution in step S910 is the same as that in Example 1, and will not be repeated here.

[0209] It should be noted that in step S910, the concentration of the regenerated silk fibroin solution is 5%–40%, and the concentration of the gelatin solution is 5%–40%. Preferably, the concentration of the regenerated silk fibroin solution is 10%, and the concentration of the gelatin solution is 30%.

[0210] As shown in Figure 10, step S910 includes:

[0211] Step S1010: Add horseradish peroxidase and hydrogen peroxide aqueous solution to the regenerated silk fibroin solution to obtain a silk fibroin composite solution.

[0212] Step S1020, the silk fibroin composite solution is mechanically shaken, so that the silk fibroin composite solution is uniformly mixed.

[0213] It should be noted that the concentration of the hydrogen peroxide aqueous solution in step S1010 is 0.1% to 1.0%, and the dosage of the hydrogen peroxide aqueous solution is 500 to 1000 μl. Preferably, the concentration of the hydrogen peroxide aqueous solution is 0.5%, and the dosage of the hydrogen peroxide aqueous solution is 800 μl.

[0214] It should be noted that the dosage of the horseradish peroxidase in step S1010 is 500-1000 μl, and the enzyme activity of the horseradish peroxidase is 600-1000 U / μl. Preferably, the dosage of the horseradish peroxidase is 700 μl, and the enzyme activity of the horseradish peroxidase is 800 U / μl.

[0215] It should be noted that the bubble removal treatment step of step S920 is the same as the bubble removal treatment step of step S110 of embodiment 1, and will not be repeated here. That is, any one of the centrifugal treatment method, the ultrasonic treatment method, the constant pressure treatment method, the vacuum treatment method, the repeated freeze-thaw treatment method, a combination of any two, a combination of any three.

[0216] The present application has the following advantages:

[0217] 1. The raw materials used in the present application, silk fibroin and gelatin, are abundant in production, low in price, and are certified by the US Food and Drug Administration as medical product raw materials, with good biocompatibility, biological function and biodegradability.

[0218] 2. The horseradish peroxidase and hydrogen peroxide used in the present application have been commercialized and produced on a large scale. Among them, low concentration of horseradish peroxidase and hydrogen peroxide has no toxicity and good biocompatibility.

[0219] 3. The silk fibroin / gelatin composite solution prepared in the present application is easy to mix uniformly during preparation, and bubbles can be effectively removed by centrifugal treatment to obtain a high-quality composite solution. This good processing performance makes the material more flexible and convenient in subsequent molding, processing and modification.

[0220] 4. The present application introduces cross-linking agents such as horseradish peroxidase and hydrogen peroxide to further promote the cross-linking reaction between silk fibroin and gelatin, forming a more compact network structure. This cross-linking reaction not only enhances the mechanical properties of the material, but also may endow the material with more functional properties, such as antibacterial, antioxidant, etc.

[0221] 5、The application can realize the accurate control of the degradation rate of the material by adjusting the ratio of silk fibroin and gelatin and the preparation process. The controllable degradation enables the material to gradually degrade and be absorbed by the human body within a predetermined time according to the specific application requirements, avoiding the need for secondary surgery.

[0222] 6、The preparation method of the application is simple and efficient, waste-free and pollution-free throughout, has good repeatability, and has potential value for industrial production.

[0223] Embodiment 3

[0224] This embodiment is a variant of embodiment 2, and the main difference between this embodiment and embodiment 2 is that step S910 is different.

[0225] As shown in FIG. 11, step S910 includes:

[0226] Step S1110, mixing the regenerated silk fibroin solution and the gelatin solution, and sequentially adding the hydrogen peroxide aqueous solution, the transglutaminase, and the horseradish peroxidase to obtain a silk fibroin composite solution;

[0227] Step S1120, mechanically shaking the silk fibroin composite solution to make the silk fibroin composite solution uniformly mixed.

[0228] It should be noted that the concentration of the regenerated silk fibroin solution, the concentration of the gelatin solution, the concentration and dosage of the hydrogen peroxide aqueous solution, and the dosage and enzyme activity of the horseradish peroxidase in step S1110 are the same as those in embodiment 2, and will not be described here.

[0229] It should be noted that the dosage of the transglutaminase in step S1110 is 10-200 μl, and the enzyme activity of the transglutaminase is 10-100 U / μl. Preferably, the dosage of the transglutaminase is 100 μl, and the enzyme activity of the transglutaminase is 50 U / μl.

[0230] The application has the following advantages:

[0231] 1、The horseradish peroxidase, hydrogen peroxide, and transglutaminase used in the application have been commercially mass-produced. Among them, the transglutaminase is prepared from microorganisms, which is more uniform in stability and abundant in yield than the original mammalian source. Low-concentration horseradish peroxidase and hydrogen peroxide have no toxicity and good biocompatibility. Experiments and literature have proved that the silk protein and gelatin hydrogel crosslinked by horseradish peroxidase and transglutaminase have no cytotoxicity and animal toxicity.

[0232] 2、The application uses horseradish peroxidase / hydrogen peroxide to induce cross-linking between silk fibroin itself and silk fibroin-gelatin, uses glutamine transaminase to modify gelatin side branches, and further induces the formation of small and uniform silk fibroin beta folds and strengthens the modification of gelatin branched chains by sequentially immersing the obtained hydrogel in 70% alcohol solution and 15% ammonium sulfate solution, so that the composite material has high strength and compression resistance, and obtains stability in simulation in vivo and in vivo.

[0233] Example 4

[0234] This embodiment relates to one specific implementation of a preparation method of a silk fibroin hard material.

[0235] A preparation method of a silk fibroin hard material, comprising:

[0236] Cut the cocoon, put it into a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution, boil for degumming treatment, and obtain silk;

[0237] Wash the silk with deionized water repeatedly, and dry the silk fibers at 40℃ for 24h to obtain the silk fibers;

[0238] Put the silk fibers into a 9M concentration lithium bromide solution for dissolution, and filter to obtain a silk fibroin stock solution;

[0239] Dialyze and desalt the silk fibroin stock solution under the condition of 10000Da to obtain a silk fibroin solution;

[0240] After reverse dialysis and concentration of the silk fibroin solution using polyethylene glycol with a concentration of 15% and a molecular weight of 20000Da, a regenerated silk fibroin solution with a concentration of 10% is obtained;

[0241] Add 700μl of horseradish peroxidase with an enzyme activity of 500U / mL and 700μl of hydrogen peroxide solution with a concentration of 0.5% to 20ml of the regenerated silk fibroin solution, and mix uniformly to obtain a regenerated silk fibroin composite solution;

[0242] Put the regenerated silk fibroin composite solution into a centrifuge tube, and place it in a centrifuge for centrifugal treatment at a centrifugal speed of 8000rpm and a centrifugal time of 30min to remove the bubbles in the regenerated silk fibroin composite solution, and obtain a regenerated silk fibroin / gel composite solution;

[0243] Place the regenerated silk fibroin / gel composite solution into a mold, and place the mold in a water bath at a water bath temperature of 35℃ for 12h to gel the regenerated silk fibroin / gel composite solution, and obtain a gel block;

[0244] The gel block is placed in a methanol solution or an ethanol solution for 30 h to induce crystallization of the gel block, to obtain a regenerated silk fibroin hard gel block;

[0245] The regenerated silk fibroin hard gel block is taken out of the methanol solution or the ethanol solution and dried to obtain a regenerated silk fibroin hard material.

[0246] Embodiment 5

[0247] This embodiment relates to one specific implementation of a method for preparing a silk fibroin hard material.

[0248] A method for preparing a silk fibroin hard material, comprising:

[0249] The cocoon is cut into pieces and boiled in a 0.02 M sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained;

[0250] The silk is repeatedly washed with deionized water and dried at 40°C for 24 h to obtain silk fibers;

[0251] The silk fibers are placed in a 9 M lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0252] The silk fibroin stock solution is dialyzed and desalted under the condition of 10,000 Da to obtain a silk fibroin solution;

[0253] The silk fibroin solution is reverse-dialyzed and concentrated using polyethylene glycol with a concentration of 15% and a molecular weight of 20,000 Da, to obtain a regenerated silk fibroin solution with a concentration of 10%;

[0254] In 20 ml of the regenerated silk fibroin solution, 700 μl of horseradish peroxidase with an enzyme activity of 500 U / mL and 700 μl of a hydrogen peroxide aqueous solution with a concentration of 0.5% are added in a dose, and mixed uniformly to obtain a regenerated silk fibroin composite solution;

[0255] The regenerated silk fibroin composite solution is horizontally flowed into a mold, and the mold is placed in an ultrasonic machine for ultrasonic treatment at an ultrasonic frequency of 50 Hz to remove air bubbles in the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained;

[0256] The regenerated silk fibroin / gel composite solution is horizontally flowed into a mold, and the mold is placed in a water bath at a water bath temperature of 35°C for 12 h to gel the regenerated silk fibroin / gel composite solution, to obtain a gel block;

[0257] The gel block is placed in a methanol solution or an ethanol solution for 30 h to induce crystallization of the gel block, to obtain a regenerated silk fibroin hard gel block;

[0258] The regenerated silk fibroin hard gel block is taken out from the methanol solution or ethanol solution and dried to obtain the regenerated silk fibroin hard material.

[0259] Example 6

[0260] This example relates to one specific embodiment of the method for preparing the silk fibroin hard material.

[0261] A method for preparing a silk fibroin hard material, comprising:

[0262] The cocoon is cut into pieces and boiled in a 0.02M sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained;

[0263] The silk is repeatedly washed with deionized water and dried at 40°C for 24h to obtain silk fibers;

[0264] The silk fibers are placed in a 9M lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0265] The silk fibroin stock solution is dialyzed and desalted under the condition of 10000Da to obtain a silk fibroin solution;

[0266] The silk fibroin solution is reverse-dialyzed and concentrated using polyethylene glycol with a concentration of 15% and a molecular weight of 20000Da, and a 10% regenerated silk fibroin solution is obtained;

[0267] In 20ml of the regenerated silk fibroin solution, 700μl of horseradish peroxidase with an enzyme activity of 500U / mL and 700μl of hydrogen peroxide aqueous solution with a concentration of 0.5% are added, and mixed uniformly to obtain a regenerated silk fibroin composite solution;

[0268] The regenerated silk fibroin composite solution is horizontally flowed into the mold, and a weight is placed on the panel of the mold to extrude the regenerated silk fibroin composite solution in the mold for 120min to remove the air bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained;

[0269] The regenerated silk fibroin / gel composite solution is horizontally flowed into the mold, and the mold is placed in a water bath at a water bath temperature of 35°C for 12h to gel the regenerated silk fibroin / gel composite solution, and a gel block is obtained;

[0270] The gel block is soaked in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and a regenerated silk fibroin hard gel block is obtained;

[0271] The regenerated silk fibroin hard gel block is taken out from the methanol solution or ethanol solution and dried to obtain the regenerated silk fibroin hard material.

[0272] Example 7

[0273] This embodiment relates to one specific implementation of a method for preparing a silk fibroin hard material.

[0274] A method for preparing a silk fibroin hard material, comprising:

[0275] Cut the cocoon into pieces, and boil in a 0.02M sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and obtain silk;

[0276] Wash the silk repeatedly with deionized water, and dry at 40°C for 24h to obtain silk fibers;

[0277] Dissolve the silk fibers in a 9M lithium bromide solution, and filter to obtain a silk fibroin stock solution;

[0278] Dialyze and desalt the silk fibroin stock solution under 10000Da conditions to obtain a silk fibroin solution;

[0279] Concentrate the silk fibroin solution by reverse dialysis using a 15% concentration and 20000Da molecular weight polyethylene glycol, and obtain a 10% regenerated silk fibroin solution;

[0280] Add 700μl of horseradish peroxidase with an enzyme activity of 500U / mL and 700μl of a 0.5% concentration hydrogen peroxide aqueous solution to 20ml of the regenerated silk fibroin solution, and mix uniformly to obtain a regenerated silk fibroin composite solution;

[0281] Lay the regenerated silk fibroin composite solution into a mold, and place the mold in a vacuum environment with a vacuum degree of 0.2MPa for 120min to remove air bubbles inside the regenerated silk fibroin composite solution, and obtain a regenerated silk fibroin / gel composite solution;

[0282] Lay the regenerated silk fibroin / gel composite solution into a mold, and place the mold in a water bath at a water bath temperature of 35°C for 12h to gel the regenerated silk fibroin / gel composite solution, and obtain a gel block;

[0283] Soak the gel block in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and obtain a regenerated silk fibroin hard gel block;

[0284] Take out the regenerated silk fibroin hard gel block from the methanol solution or the ethanol solution, and dry to obtain a regenerated silk fibroin hard material.

[0285] Example 8

[0286] This embodiment relates to one specific implementation of a method for preparing a silk fibroin hard material.

[0287] A method for preparing a silk fibroin hard material, comprising:

[0288] Cutting the cocoon into pieces, and boiling in a 0.02M sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and obtaining silk;

[0289] Washing the silk repeatedly with deionized water, and drying at 40℃ for 24h to obtain silk fibers;

[0290] Dissolving the silk fibers in a 9M lithium bromide solution, and filtering to obtain a silk fibroin stock solution;

[0291] Dialyzing and desalting the silk fibroin stock solution under 10000Da conditions to obtain a silk fibroin solution;

[0292] Reverse dialyzing and concentrating the silk fibroin solution using a 15% concentration, 20000Da molecular weight polyethylene glycol to obtain a 10% concentration regenerated silk fibroin solution;

[0293] Adding 700μl of a 500U / mL enzyme activity horseradish peroxidase and 700μl of a 0.5% concentration hydrogen peroxide aqueous solution to 20ml of the regenerated silk fibroin solution, and mixing uniformly to obtain a regenerated silk fibroin composite solution;

[0294] Flowing the regenerated silk fibroin composite solution into a mold, and placing the mold in a low temperature environment of -60℃ for 5 days to freeze the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin composite solution frozen block, and dissolving the regenerated silk fibroin composite solution frozen block to obtain a regenerated silk fibroin composite solution, repeating the above steps 2 times to remove air bubbles in the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin / gel composite solution;

[0295] Flowing the regenerated silk fibroin / gel composite solution into a mold, and placing the mold in a water bath at a water bath temperature of 35℃ for 12h to gel the regenerated silk fibroin / gel composite solution, and obtaining a gel block;

[0296] Soaking the gel block in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and obtaining a regenerated silk fibroin hard gel block;

[0297] Taking the regenerated silk fibroin hard gel block out of the methanol solution or the ethanol solution, and drying to obtain a regenerated silk fibroin hard material.

[0298] Example 9

[0299] This example relates to one specific embodiment of a method for preparing a silk fibroin hard material.

[0300] A method for preparing a silk fibroin hard material, comprising:

[0301] Cutting the cocoon and placing it in a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for boiling degumming treatment, and obtaining silk;

[0302] Washing the silk repeatedly with deionized water and drying it at 40°C for 24h to obtain silk fibers;

[0303] Dissolving the silk fibers in a 9M concentration lithium bromide solution and filtering to obtain a silk fibroin stock solution;

[0304] Dialyzing and desalting the silk fibroin stock solution under 10000Da conditions to obtain a silk fibroin solution;

[0305] After reverse dialysis and concentration of the silk fibroin solution using a 15% concentration, 20000Da molecular weight polyethylene glycol, a 10% concentration regenerated silk fibroin solution is obtained;

[0306] Mixing the 10% concentration regenerated silk fibroin solution with a 30% concentration gelatin solution, and sequentially adding a 0.5% concentration, 700μl dose hydrogen peroxide aqueous solution, a 100μl dose, 50U / μl enzyme activity transglutaminase, a 700μl dose, 500U / mL enzyme activity horseradish peroxidase, and mixing uniformly to obtain a regenerated silk fibroin composite solution;

[0307] Placing the regenerated silk fibroin composite solution in a centrifuge tube and placing it in a centrifuge for centrifugal treatment at a centrifugal speed of 8000rpm and a centrifugal time of 30min to remove air bubbles inside the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin / gel composite solution;

[0308] Laminating the regenerated silk fibroin / gel composite solution into a mold, and placing the mold in a water bath at a water bath temperature of 35°C for 12h to gel the regenerated silk fibroin / gel composite solution, and obtaining a gel block;

[0309] Soaking the gel block in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and obtaining a regenerated silk fibroin hard gel block;

[0310] Taking the regenerated silk fibroin hard gel block out of the methanol solution or the ethanol solution and drying it to obtain a regenerated silk fibroin hard material.

[0311] Example 10

[0312] This example relates to one specific embodiment of a method for preparing a silk fibroin hard material.

[0313] A method for preparing a silk fibroin hard material, comprising:

[0314] Cutting the cocoon, putting it into a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for boiling and degumming treatment, and obtaining silk;

[0315] Washing the silk repeatedly with deionized water, and drying it at 40℃ for 24h to obtain silk fiber;

[0316] Putting the silk fiber into a 9M concentration lithium bromide solution for dissolution, and filtering to obtain a silk fibroin stock solution;

[0317] Dialyzing and desalting the silk fibroin stock solution under the condition of 10000Da to obtain a silk fibroin solution;

[0318] After reverse dialysis and concentration of the silk fibroin solution using polyethylene glycol with a concentration of 15% and a molecular weight of 20000Da, a 10% regenerated silk fibroin solution is obtained;

[0319] Mixing the 10% regenerated silk fibroin solution with a 30% gelatin solution, and sequentially adding 0.5% concentration, 700μl dosage hydrogen peroxide aqueous solution, 100μl dosage, 50U / μl enzyme activity transglutaminase, 700μl dosage, 500U / mL enzyme activity horseradish peroxidase, and mixing uniformly to obtain a regenerated silk fibroin composite solution;

[0320] Making the silk fibroin composite solution flow horizontally into a mold, and placing the mold in an ultrasonic machine for ultrasonic treatment at an ultrasonic frequency of 50Hz to remove air bubbles inside the silk fibroin composite solution, and obtaining a silk fibroin / gel composite solution;

[0321] Making the silk fibroin / gel composite solution flow horizontally into a mold, and placing the mold in a water bath at a water bath temperature of 35℃ for 12h to gel the silk fibroin / gel composite solution, and obtaining a gel block;

[0322] Placing the gel block in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and obtaining a silk fibroin hard material;

[0323] Taking the silk fibroin hard material out of the methanol solution or the ethanol solution, and freeze-drying it for 5 days to obtain a regenerated silk fibroin hard material.

[0324] Example 11

[0325] This example relates to one specific embodiment of a method for preparing a silk fibroin hard material.

[0326] A method for preparing a silk fibroin hard material, comprising:

[0327] Cut the cocoon into pieces, and put them into a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for boiling and degumming treatment, and obtain silk;

[0328] Wash the silk repeatedly with deionized water, and dry it at 40℃ for 24h to obtain silk fibers;

[0329] Put the silk fibers into a 9M concentration lithium bromide solution for dissolution, and filter to obtain a silk fibroin stock solution;

[0330] Dialyze and desalt the silk fibroin stock solution under the condition of 10000Da to obtain a silk fibroin solution;

[0331] Concentrate the silk fibroin solution by reverse dialysis using 15% concentration and 20000Da molecular weight polyethylene glycol, and obtain a 10% concentration regenerated silk fibroin solution;

[0332] Mix the 10% concentration regenerated silk fibroin solution with a 30% concentration gelatin solution, and sequentially add 0.5% concentration, 700μl dosage hydrogen peroxide aqueous solution, 100μl dosage, 50U / μl enzyme activity transglutaminase, 700μl dosage, 500U / mL enzyme activity horseradish peroxidase, and mix uniformly to obtain a regenerated silk fibroin composite solution;

[0333] Lay the regenerated silk fibroin composite solution to the mold, and place a weight on the panel of the mold to extrude the silk fibroin composite solution in the mold for 120min to remove the air bubbles inside the regenerated silk fibroin composite solution, and obtain a regenerated silk fibroin / gel composite solution;

[0334] Lay the regenerated silk fibroin / gel composite solution to the mold, and place the mold in a water bath at a water bath temperature of 35℃ for 12h to gel the regenerated silk fibroin / gel composite solution, and obtain a gel block;

[0335] Soak the gel block in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and obtain a regenerated silk fibroin hard gel block;

[0336] Take out the regenerated silk fibroin hard gel block from the methanol solution or the ethanol solution, and freeze-dry it for 5 days to obtain a regenerated silk fibroin hard material.

[0337] Example 12

[0338] This example relates to one specific embodiment of a method for preparing a silk fibroin hard material.

[0339] A method for preparing a silk fibroin hard material, comprising:

[0340] The cocoon is cut into pieces and put into a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for boiling degumming treatment, and silk is obtained;

[0341] The silk is repeatedly washed with deionized water and dried at 40°C for 24h to obtain silk fibers;

[0342] The silk fibers are put into a 9M concentration lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0343] The silk fibroin stock solution is dialyzed and desalted under the condition of 10000Da to obtain a silk fibroin solution;

[0344] The silk fibroin solution is reverse dialyzed and concentrated using 15% concentration and 20000Da molecular weight polyethylene glycol, and a 10% concentration regenerated silk fibroin solution is obtained;

[0345] The 10% concentration regenerated silk fibroin solution is mixed with a 30% concentration gelatin solution, and 0.5% concentration hydrogen peroxide solution with a dose of 700μl, 100μl transglutaminase with an enzyme activity of 50U / μl, 700μl horseradish peroxidase with an enzyme activity of 500U / mL are sequentially added and uniformly mixed to obtain a regenerated silk fibroin composite solution;

[0346] The regenerated silk fibroin composite solution is horizontally flowed into a mold, and the mold is placed in a vacuum environment with a vacuum degree of 0.2MPa for 120min to remove the bubbles inside the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained;

[0347] The regenerated silk fibroin / gel composite solution is horizontally flowed into a mold, and the mold is placed in a water bath environment with a water bath temperature of 35°C for 12h to gel the regenerated silk fibroin / gel composite solution, and a gel block is obtained;

[0348] The gel block is soaked in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and a regenerated silk fibroin hard gel block is obtained;

[0349] The regenerated silk fibroin hard gel block is taken out of the methanol solution or the ethanol solution and freeze-dried for 5 days to obtain a regenerated silk fibroin hard material.

[0350] Example 13

[0351] This example relates to one specific embodiment of a method for preparing a silk fibroin hard material.

[0352] A method for preparing a silk fibroin hard material, comprising:

[0353] The cocoon is cut into pieces and put into a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for boiling degumming treatment, and silk is obtained;

[0354] The silk is repeatedly washed with deionized water and dried at 40°C for 24h to obtain silk fibers;

[0355] The silk fibers are put into a 9M concentration lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0356] The silk fibroin stock solution is dialyzed and desalted under the condition of 10000Da to obtain a silk fibroin solution;

[0357] The silk fibroin solution is reverse dialyzed and concentrated using 15% concentration and 20000Da molecular weight polyethylene glycol to obtain a 10% concentration regenerated silk fibroin solution;

[0358] The 10% concentration regenerated silk fibroin solution is mixed with a 30% concentration gelatin solution, and 0.5% concentration hydrogen peroxide solution with a dose of 700μl, 100μl transglutaminase with an enzyme activity of 50U / μl, 700μl horseradish peroxidase with an enzyme activity of 500U / mL are sequentially added and uniformly mixed to obtain a regenerated silk fibroin composite solution;

[0359] The regenerated silk fibroin composite solution is horizontally flowed into a mold, and the mold is placed in a low-temperature environment of-60°C for freezing for 5 days to freeze the regenerated silk fibroin composite solution, and a regenerated silk fibroin composite solution frozen block is obtained. The composite solution frozen block is dissolved to obtain a regenerated silk fibroin composite solution. The above steps are repeated twice to remove the air bubbles in the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained;

[0360] The regenerated silk fibroin / gel composite solution is horizontally flowed into a mold, and the mold is placed in a water bath environment with a water bath temperature of 35°C for 12h to gel the regenerated silk fibroin / gel composite solution to obtain a gel block;

[0361] The gel block is soaked in a methanol solution or an ethanol solution for 30h to induce crystallization of the gel block, and a regenerated silk fibroin hard gel block is obtained;

[0362] The regenerated silk fibroin hard gel block is taken out of the methanol solution or the ethanol solution and freeze-dried for 5 days to obtain a regenerated silk fibroin hard material.

[0363] Example 14

[0364] The sample was prepared according to the method described in Embodiment 4 to Embodiment 13, and the states before and after removing the bubbles were shown in FIG. 12 and FIG. 13, respectively. In FIG. 12, FIG. 12a is the state of the gel containing bubbles, FIG. 12b is the state of the gel after removing the bubbles by ultrasonic treatment, FIG. 12c is the state of the rod-shaped embryo material containing bubbles after solidification of FIG. 12a, and FIG. 12d is the state of the rod-shaped embryo material without bubbles after solidification of FIG. 12b. In FIG. 13, FIG. 13a is the state of the gel containing bubbles in a centrifugal tube, FIG. 13b is the state of the gel after removing the bubbles by centrifugal separation treatment, and FIG. 13c is the state of the embryo material without bubbles after solidification of FIG. 13b. The other embodiments in the present scheme can also achieve the effect of removing bubbles as shown in FIG. 12d and FIG. 13c.

[0365] The compression resistance of the silk fibroin solid embryo material without bubbles in FIG. 12d and FIG. 13c and the silk fibroin solid embryo material with bubbles in FIG. 12c was tested according to the following method.

[0366] According to the test method of 4.6 mechanical strength in the Chinese national industry recommended standard YY / T 1558.3, a universal tensile testing machine was used, and a metal pressing module and a base were customized. The sample was cut into a cylindrical sample block with a diameter of 4 mm and a length of 2 cm, and was placed in the center of the metal base, and the pressing module was fixed above it. The universal tensile testing machine was used to apply a constant speed pressure to the sample block, and the speed was 0.5±0.05 mm / min. The test was stopped when the sample block was cracked, and the maximum force value (unit: N) was recorded.

[0367] The results are shown in FIG. 14. The test showed that the sample block with bubbles collapsed locally due to internal defects when subjected to pressure, and the pressure curve showed a broken line trend. The pressure curve of the sample block without bubbles was a straight line, and the maximum force value was greater than that of the sample block with bubbles. Through the test, the maximum compression strength of the sample block after removing the bubbles by ultrasonic method in Embodiment 5 was 81.7±2.8 Mpa, and the maximum compression strength of the sample with bubbles was 64.5±4.5 Mpa, and the statistical p value was <0.05. The sample block after removing the bubbles was significantly better than the sample with bubbles in mechanical properties.

[0368] Embodiment 15

[0369] An illustrative embodiment of the present application is shown in FIG. 15, a method for preparing a composite bone repair material, comprising:

[0370] In step S1510, the silkworm cocoons are immersed in a soap salt solution for degumming, dried to prepare degummed silk, and then dissolved in a lithium bromide solution, dialyzed to remove salt, and concentrated to obtain a regenerated silk fibroin solution.

[0371] Step S1520, mix the regenerated silk fibroin solution with the gelatin solution, and sequentially add the hydrogen peroxide aqueous solution, the transglutaminase, and the horseradish peroxidase to obtain a gel solution;

[0372] Step S1530, mechanically shake the gel solution to make the gel solution mixed uniformly and form bubbles uniformly distributed in the gel solution, and then horizontally flow the uniformly mixed gelatin solution to the mold to gel, and obtain a regenerated silk fibroin / gelatin gel block;

[0373] Step S1540, soak and clean the regenerated silk fibroin / gelatin gel block in the ammonium sulfate solution to obtain a regenerated silk fibroin / gelatin cross-linked gel;

[0374] Step S1550, freeze-dry the regenerated silk fibroin / gelatin cross-linked gel to obtain a regenerated silk fibroin / gelatin composite material.

[0375] It should be noted that the concentration of the regenerated silk fibroin solution in step S1520 is 5% to 40%, and the concentration of the gelatin solution is 5% to 40%. Preferably, the concentration of the regenerated silk fibroin solution is 10%, and the concentration of the gelatin solution is 30%.

[0376] It should be noted that the concentration of the hydrogen peroxide aqueous solution in step S1520 is 0.1% to 1.0%, and the dosage of the hydrogen peroxide aqueous solution is 500 to 1000 μl. Preferably, the concentration of the hydrogen peroxide aqueous solution is 0.5%, and the dosage of the hydrogen peroxide aqueous solution is 800 μl.

[0377] It should be noted that the dosage of the transglutaminase in step S1520 is 10 to 200 μl, and the enzyme activity of the transglutaminase is 10 to 100 U / μl. Preferably, the dosage of the transglutaminase is 100 μl, and the enzyme activity of the transglutaminase is 50 U / μl.

[0378] It should be noted that the dosage of the horseradish peroxidase in step S1520 is 500-1000 μl, and the enzyme activity of the horseradish peroxidase is 600-1000 U / mL. Preferably, the dosage of the horseradish peroxidase is 700 μl, and the enzyme activity of the horseradish peroxidase is 800 U / mL.

[0379] It should be noted that the concentration of the ammonium sulfate solution in step S1540 is 10% to 20%, and the dosage of the ammonium sulfate solution is 100 to 200 ml. Preferably, the concentration of the ammonium sulfate solution is 15%, and the dosage of the ammonium sulfate solution is 150 ml (generally 20 ml of gel needs 100-200 ml of ammonium sulfate solution).

[0380] It should be noted that the freezing drying time in step S1550 is 2-8 days. Preferably, the freezing drying time is 5 days.

[0381] As shown in FIG. 16, step S1510 includes:

[0382] Step S1610, the cocoon is cut into pieces, boiled in sodium carbonate-sodium bicarbonate buffer for degumming treatment, and silk is obtained;

[0383] Step S1620, the silk is repeatedly washed with deionized water, and dried to obtain silk fibers;

[0384] Step S1630, the silk fibers are placed in a lithium bromide solution for dissolution, and filtered to obtain a silk fibroin stock solution;

[0385] Step S1640, the silk fibroin stock solution is dialyzed and desalted to obtain a silk fibroin solution;

[0386] Step S1650, the silk fibroin solution is reverse-dialyzed and concentrated using polyethylene glycol to obtain a regenerated silk fibroin solution.

[0387] It should be noted that the concentration of the sodium carbonate-sodium bicarbonate buffer in step S1610 is 0.01M-0.05M. Preferably, the concentration of the sodium carbonate-sodium bicarbonate buffer is 0.02M.

[0388] It should be noted that the drying temperature in step S1620 is 35°C-45°C, and the drying time is 20h-30h. Preferably, the drying temperature is 340°C, and the drying time is 24h.

[0389] It should be noted that the concentration of the lithium bromide solution in step S1630 is 8-10M. Preferably, the concentration of the lithium bromide solution is 9M.

[0390] It should be noted that the dialysis molecular weight in step S1640 is 8000Da-14000Da. Preferably, the dialysis molecular weight is 10000Da.

[0391] It should be noted that the molecular weight of the polyethylene glycol in step S1650 is 10000Da-30000Da, and the concentration of the polyethylene glycol is 10%-20%. Preferably, the molecular weight of the polyethylene glycol is 20000Da, and the concentration of the polyethylene glycol is 15%.

[0392] It should be noted that the concentration of the regenerated silk fibroin solution in step S1650 is 5%-20%. Preferably, the concentration of the regenerated silk fibroin solution is 10%.

[0393] As shown in FIG. 17, step S1530 includes:

[0394] Step S1710, the gel solution is mechanically shaken, so that the gel solution is mixed uniformly and the bubbles are uniformly distributed in the gel solution, and the uniformly mixed gelatin solution is horizontally flowed into the mold to gel;

[0395] Step S1720, after the preliminary gelation, the gel is cut, and a plurality of regenerated silk fibroin / gelatin gel blocks are obtained.

[0396] It should be noted that the mechanical shaking measure in step S1710 can be performed by vortexing at different speeds, repeated rotation, stirring, stirring paddle stirring, etc.

[0397] It should be noted that the size of the silk fibroin / gelatin gel block in step S1710 is 2-4mm 3 . Preferably, the size of the regenerated silk fibroin / gelatin gel block is 3mm 3 .

[0398] It should be noted that the shape of the composite bone repair material is set as regular particles, irregular particles, spherical solids, cylindrical solids, cubic solids, rectangular solids, irregular shape solids.

[0399] It should be noted that the composite bone repair material is set as a porous structure in micrometer or nanometer scale, and the porosity of the composite bone repair material is 20%-90%.

[0400] It should be noted that the structural strength (compression strength) of the composite bone repair material is 0.1-35MPa.

[0401] It should be noted that the composite bone repair material is applied to the preparation of bone filling material, and is applied to the filling and regenerative repair of bone defects in orthopedics, oral surgery, plastic surgery, neurosurgery skull.

[0402] The present application has the following advantages:

[0403] 1. The raw materials silk fibroin and gelatin used in the present application have rich yield, low price and are certified medical product raw materials by the US Food and Drug Administration, and have good biocompatibility, biological function and biodegradability;

[0404] 2. The horseradish peroxidase, hydrogen peroxide and glutamine transaminase used in the present application have realized commercial large-scale production. The glutamine enzyme is prepared from microorganisms, which has more uniform stability and rich yield than the original mammalian source. Low concentration of horseradish peroxidase and hydrogen peroxide has no toxicity and good biocompatibility. Experiments and literature have proved that the silk fibroin and gelatin hydrogel crosslinked by horseradish peroxidase and glutamine transaminase has no cell and animal toxicity;

[0405] 3、The application applies horseradish peroxidase / hydrogen peroxide to induce crosslinking between silk fibroin itself and silk fibroin-gelatin, applies glutamine transaminase to modify gelatin side branches, and further induces the formation of small and uniform silk fibroin beta folds and strengthens the modification of gelatin branched chains by sequentially immersing the obtained hydrogel in 70% alcohol solution and 15% ammonium sulfate solution, so that the composite material has high strength and compression resistance and obtains stability in simulation in vivo and in vivo;

[0406] 4、The application has the biological functions of improving cell adhesion, growth and differentiation by adding gelatin on the basis of the existing silk fibroin material;

[0407] 5、The application prepares a porous material on the basis of the existing silk fibroin / gelatin material, has the advantages of high porosity, and greatly promotes cell growth, such as cell proliferation and migration, and tissue growth, which is very beneficial to the clinical application of tissue engineering technology;

[0408] 6、The composite material prepared by the application can provide a three-dimensional space for the adhesion, proliferation and migration of bone repair cells, and nutrients and moisture can also be transported to the cells through the pores; and the bone filling material prepared by compounding gelatin and silk fibroin can enhance the osteogenic activity of the material;

[0409] 7、The preparation method of the application is simple and efficient, has no waste and pollution in the whole process, has good repeatability, and has potential value for industrial production.

[0410] Example 16

[0411] This embodiment relates to one specific embodiment of the preparation method of the composite bone repair material.

[0412] The preparation method of the composite bone repair material comprises the following steps:

[0413] The cocoon is cut into pieces, boiled in a 0.02M concentration sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained;

[0414] The silk is repeatedly washed with deionized water and dried at 40℃ for 24h to obtain silk fibers;

[0415] The silk fibers are dissolved in a 9M concentration lithium bromide solution, and filtered to obtain a silk fibroin stock solution;

[0416] The silk fibroin stock solution is dialyzed and desalted under the condition of 10000Da to obtain a silk fibroin solution;

[0417] The silk fibroin solution is reverse-dialyzed and concentrated by using polyethylene glycol with a concentration of 15% and a molecular weight of 20000Da, and a 10% regenerated silk fibroin solution is obtained.

[0418] The 10% concentration regenerated silk fibroin solution and the 30% concentration gelatin solution are uniformly mixed in a ratio of 85:15, and then 0.5% concentration hydrogen peroxide solution with a dosage of 800 μl, 100 μl of transglutaminase with an enzyme activity of 50 U / μl, and 700 μl of horseradish peroxidase with an enzyme activity of 500 U / mL are sequentially added to obtain a regenerated silk fibroin composite solution;

[0419] The regenerated silk fibroin composite solution is mechanically shaken to uniformly mix the regenerated silk fibroin composite solution and form uniformly distributed bubbles in the regenerated silk fibroin composite solution, and the uniformly mixed regenerated silk fibroin composite solution is horizontally flowed into the mold to gel;

[0420] After the initial gelation, the gel is cut to obtain several regenerated silk fibroin / gelatin gel blocks with a size of 3 mm 3 ;

[0421] The regenerated silk fibroin / gelatin gel blocks are soaked and washed in a 15% concentration ammonium sulfate solution to obtain regenerated silk fibroin / gelatin crosslinked gel.

[0422] The regenerated silk fibroin / gelatin crosslinked gel is freeze-dried for 5 days to obtain a regenerated silk fibroin / gelatin composite bone repair material.

[0423] The advantages of this embodiment are the same as those of Example 15, which will not be repeated here.

[0424] Example 17

[0425] This embodiment relates to another specific embodiment of the preparation method of the composite bone repair material.

[0426] A preparation method of a composite bone repair material, comprising:

[0427] The cocoon is cut into pieces, boiled in a 0.02 M concentration sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and silk is obtained.

[0428] The silk is repeatedly washed with deionized water and dried at 40°C for 24 h to obtain silk fibers.

[0429] The silk fibers are dissolved in a 9 M concentration lithium bromide solution and filtered to obtain a silk fibroin stock solution.

[0430] The silk fibroin stock solution is dialyzed and desalted under the condition of 10,000 Da to obtain a silk fibroin solution.

[0431] The silk fibroin solution is reverse-dialyzed and concentrated using 15% concentration polyethylene glycol with a molecular weight of 20,000 Da to obtain a 10% concentration regenerated silk fibroin solution.

[0432] The 10% concentration regenerated silk fibroin solution is mixed with the 30% concentration gelatin solution at a ratio of 60:40, and then 0.5% concentration hydrogen peroxide solution with a dosage of 800 μl, 100 μl transglutaminase with an enzyme activity of 50 U / μl, and 700 μl horseradish peroxidase with an enzyme activity of 500 U / mL are sequentially added to obtain a regenerated silk fibroin composite solution;

[0433] The regenerated silk fibroin composite solution is mechanically shaken to mix the regenerated silk fibroin composite solution uniformly and form bubbles uniformly distributed in the regenerated silk fibroin composite solution, and the uniformly mixed regenerated silk fibroin composite solution is horizontally flowed into the mold to gel;

[0434] After the initial gelation, the gel is cut to obtain several regenerated silk fibroin / gelatin gel blocks with a size of 3 mm 3 ;

[0435] The regenerated silk fibroin / gelatin gel blocks are soaked and washed in a 15% concentration ammonium sulfate solution to obtain regenerated silk fibroin / gelatin crosslinked gel;

[0436] The regenerated silk fibroin / gelatin crosslinked gel is freeze-dried for 5 days to obtain a regenerated silk fibroin / gelatin composite bone repair material.

[0437] The advantages of this embodiment are the same as those of Example 15, which will not be repeated here.

[0438] Example 18

[0439] This embodiment relates to another specific embodiment of the preparation method of the composite bone repair material.

[0440] A preparation method of a composite bone repair material, comprising:

[0441] The cocoon is cut into pieces and boiled in a 0.02 M concentration sodium carbonate-sodium bicarbonate buffer solution for degumming treatment to obtain silk;

[0442] The silk is repeatedly washed with deionized water and dried at 40°C for 24 h to obtain silk fibers;

[0443] The silk fibers are dissolved in a 9 M concentration lithium bromide solution and filtered to obtain a silk fibroin stock solution;

[0444] The silk fibroin stock solution is dialyzed and desalted under the condition of 10,000 Da to obtain a silk fibroin solution;

[0445] The silk fibroin solution is reverse-dialyzed and concentrated using 15% concentration polyethylene glycol with a molecular weight of 20,000 Da to obtain a 10% concentration regenerated silk fibroin solution;

[0446] The 10% concentration regenerated silk fibroin solution is mixed with the 30% concentration gelatin solution at a ratio of 85:15, and then 0.5% concentration hydrogen peroxide solution with a dosage of 800 μl, 100 μl of transglutaminase with an enzyme activity of 50 U / μl, and 700 μl of horseradish peroxidase with an enzyme activity of 500 U / mL are sequentially added to obtain a regenerated silk fibroin composite solution;

[0447] The regenerated silk fibroin composite solution is mechanically shaken to mix the regenerated silk fibroin composite solution uniformly and form bubbles uniformly distributed in the regenerated silk fibroin composite solution, and the uniformly mixed regenerated silk fibroin composite solution is horizontally flowed into the mold to gel;

[0448] After the initial gelation, the gel is cut to obtain several regenerated silk fibroin / gelatin gel blocks with a size of 3 mm 3 ;

[0449] The gel blocks are soaked and washed in a 75% concentration ethanol solution to obtain regenerated silk fibroin / gelatin crosslinked gel;

[0450] The regenerated silk fibroin / gelatin gel blocks are soaked and washed in a 15% concentration ammonium sulfate solution to obtain regenerated silk fibroin / gelatin crosslinked gel;

[0451] The regenerated silk fibroin / gelatin crosslinked gel is freeze-dried for 5 days to obtain a regenerated silk fibroin / gelatin composite bone repair material.

[0452] The advantages of this embodiment are the same as those of Example 15, which will not be repeated here.

[0453] Example 19

[0454] This embodiment relates to another specific embodiment of the preparation method of the composite bone repair material.

[0455] A preparation method of a composite bone repair material, comprising:

[0456] The cocoon is cut into pieces and boiled in a 0.02 M concentration sodium carbonate-sodium bicarbonate buffer solution for degumming treatment to obtain silk;

[0457] The silk is repeatedly washed with deionized water and dried at 40°C for 24 h to obtain silk fibers;

[0458] The silk fibers are dissolved in a 9 M concentration lithium bromide solution and filtered to obtain a silk fibroin stock solution;

[0459] The silk fibroin stock solution is dialyzed and desalted under the condition of 10,000 Da to obtain a silk fibroin solution;

[0460] The silk fibroin solution is reverse-dialyzed and concentrated using polyethylene glycol with a concentration of 15% and a molecular weight of 20,000 Da, to obtain a regenerated silk fibroin solution with a concentration of 10%;

[0461] The regenerated silk fibroin solution with a concentration of 10% is mixed with a gelatin solution with a concentration of 30% at a ratio of 85:15, and then 0.5% hydrogen peroxide solution with a dosage of 800 μl, 100 μl of transglutaminase with an enzyme activity of 50 U / μl, and 700 μl of horseradish peroxidase with an enzyme activity of 500 U / mL are sequentially added to obtain a regenerated silk fibroin composite solution;

[0462] The regenerated silk fibroin composite solution is horizontally flowed into a mold to gel;

[0463] After the initial gelation, the gel is cut to obtain several regenerated silk fibroin / gelatin gel blocks with a size of 3 mm 3 ;

[0464] The regenerated silk fibroin / gelatin gel blocks are soaked and cleaned in an ammonium sulfate solution with a concentration of 15% to obtain regenerated silk fibroin / gelatin crosslinked gels;

[0465] The regenerated silk fibroin / gelatin crosslinked gels are freeze-dried for 5 days to obtain regenerated silk fibroin / gelatin composite bone repair materials.

[0466] The advantages of this embodiment are the same as those of Example 15, which will not be repeated here.

[0467] Example 20

[0468] This example relates to the porosity and pore size test of the composite bone repair material

[0469] The regenerated silk fibroin / gelatin composite bone repair materials obtained in Examples 16-19 are tested for porosity and pore size distribution according to the following method.

[0470] According to the international standardization organization (ISO) recommended standard ISO 15901-1:2005, the porosity and pore size distribution of the bone repair materials obtained in different examples are tested using a MIP mercury intrusion porosimeter, a dilatometer, and measuring liquid mercury. Several sample blocks with a size of about 3-5 mm 3 are weighed and the data is recorded; then, the samples are placed in a dry and clean sample dilatometer, the samples are vacuumed to remove water vapor and other gases, and the samples are kept in a vacuum state; finally, mercury is injected into the sample dilatometer, and the pressure is less than 5 kPa; during the pressurization process, the external pressure and the volume of mercury injection are recorded using a chart or a computer; after the test is completed, the pore size distribution and porosity are obtained by plotting the recorded values.

[0471] The experimental results are shown in Table 1. The porosities of the composite bone repair materials prepared by the preparation methods described in Examples 2, 3 and 5 are all greater than 50% and less than 75%; the pore size distributions are in the range of 1 nm to 500 μm; among them, the pore size distribution of the composite bone repair material prepared by the preparation method described in Example 2 is narrower, in the range of 1 μm to 200 μm. The scanning electron microscope (SEM) photograph of the composite bone repair material prepared by the preparation method described in Example 2 is shown in FIG. 18.

[0472] Example 21

[0473] This example relates to the compression strength test of the composite bone repair material

[0474] The regenerated silk fibroin / gelatin composite bone repair material obtained in Example 16 was tested for compression resistance mechanical strength according to the following method.

[0475] According to the international standardization organization (ISO) recommended standard ISO 13175-3:2012, the bone repair materials obtained in different examples were subjected to compression test using an Instron 3366 (Instron, USA) universal mechanical tester, a 10 kN load cell, a customized pressure test metal base and a pressure block (hardness of 300 HV or higher). The sample was pre-processed into a cuboid sample block of 10°10°15 mm, and was fixed at the center of the metal base; the metal pressure block was loaded on the universal mechanical tester, located directly above the sample block; the compression rate was set to 0.5 mm / min, and the sample block was compressed until the sample block was destroyed, and the maximum load was recorded and the compression strength (unit: MPa) was calculated.

[0476] The experimental results are shown in Table 1. The compression strengths of the bone repair materials of Examples 16, 18 and 19 are all greater than 2.0 MPa, and in particular, the compression strength of Example 18 can reach 3.6±0.5 MPa. In combination with the test results of Example 20, it can be seen that the preparation method of the present application can prepare bone repair materials with different porosities, pore size distributions and compression mechanical properties, which have potential clinical application prospects and values.

[0477] Table 1. Comparison of different samples of bone repair materials

[0478] Example 22

[0479] This example relates to the cell compatibility test of the composite bone repair material

[0480] The regenerated silk fibroin / gelatin composite bone repair material obtained in Example 16 was tested for cell adhesion according to the following method.

[0481] To evaluate the cell adhesion of the bone repair material, bone marrow mesenchymal stem cells (BMSCs) were seeded onto the bone repair material at a density of 1 x 104 / mL and co-cultured for 3 days. Then the samples were washed with phosphate buffer solution (PBS) to remove non-adherent cells, fixed with glutaraldehyde at 4°C, and dehydrated by gradient ethanol solution. After that, the samples were dried in a blast drying oven at 40°C for 2 days. The cell adhesion micro-SEM photos of the surface of the composite bone repair material obtained by the preparation method described in Example 2 and their enlarged pictures are shown in Figure 19.

[0482] As can be seen from the pictures, the cells have successfully adhered to the material surface, and part of the cells have entered the pores on the surface, with obvious pseudopodia visible, connected to each other (as indicated by the arrows), indicating that the silk fibroin / gelatin composite bone repair material prepared by the present preparation method has good cell compatibility, which is conducive to cell adhesion, proliferation and migration.

[0483] Example 23

[0484] This example relates to the in vitro pull-out force mechanical test of the bone pin.

[0485] The regenerated silk fibroin hard material obtained in Example 4 was processed into a bone pin using a multi-axis numerical control machining lathe, obtaining a regenerated silk fibroin bone pin (RSF). The size of the RSF bone pin is as follows: screw diameter 2 mm, screw length 5 mm, tail cap diameter 3.6 mm.

[0486] The regenerated silk fibroin / gelatin composite bone repair material obtained in Example 16 was processed into a columnar or block-shaped parent embryo, and then a multi-axis numerical control machining lathe was used to process it into a bone pin, obtaining a regenerated silk fibroin / gelatin composite bone pin (RSF / G). The size of the RSF / G bone pin is as follows: screw diameter 2 mm, screw length 5 mm, tail cap diameter 3.6 mm.

[0487] As a comparison, polylactic acid-glycolic acid copolymer (PLGA) was used as a raw material to process a bone pin of the same size using a multi-axis numerical control machining lathe.

[0488] For the above three kinds of bone pins, the pull-out force in dry state and wet state was tested according to the following method, respectively.

[0489] The pull-out strength of RSF / G bone pins, RSF bone pins and PLGA bone pins was tested according to the American Society for Testing and Materials (ASTM) standard F1839, using an Instron 5966 (Instron, USA) universal testing machine, custom-made bone pin tail cap stainless steel clamps and artificial bone blocks (Sawbones, USA). The artificial bone blocks were pre-embedded with bone cement, fixed at the bottom of the Instron 5966 testing frame and drilled with a drill bit (1.7 mm in diameter). The RSF / G bone pins and RSF bone pins were screwed into the artificial bone blocks, and then the bone pin tail caps were clamped with the clamps and connected to the Instron testing machine. The bone pins were gradually pulled out of the artificial bone blocks using the testing machine at a constant speed of 5.0 mm / min until the bone pins were pulled out or broken (ASTM standard F2502-11). When the bone pins were pulled out or broken, the maximum stress (unit: N) was recorded. The pull-out mechanical tests were performed in dry and wet states, respectively, and the wet state was prepared by soaking the bone pins in a PBS solution at 37°C for more than 72 hours.

[0490] The results are shown in Figure 20. The experiments showed that the pull-out forces of the RSF / G bone pins, RSF bone pins and PLGA bone pins were 46 N, 60 N and 49 N, respectively, in the dry state; and the pull-out forces of the RSF / G bone pins, RSF bone pins and PLGA bone pins were 26 N, 30 N and 50 N, respectively, in the wet state. It can be seen that the pull-out force of the RSF / G bone pins was slightly lower than that of the RSF bone pins and PLGA bone pins in both the dry state and the wet state, but there was no significant statistical difference.

[0491] Example 24

[0492] This example relates to the in vitro shear force mechanical test of bone pins.

[0493] The RSF / G bone pins, RSF bone pins and PLGA bone pins were prepared according to Example 23.

[0494] The double shear strength in the dry state and the wet state was tested according to the following method, respectively.

[0495] The double shear test is completed by a customized clamp and an Instron 5966 (Instron, USA) universal material testing machine. The clamp is composed of three stainless steel plates with aligned 2mm holes and 2.5mm holes respectively for shear test in dry and wet states. The RSF / G bone screws, RSF bone screws and PLGA bone screws with 2mm are made. The fixture is installed on the Instron 5966 (Instron, USA) testing frame, the bottom of the fixture remains stationary, while the top of the fixture is stretched at a speed of 5.0mm / min until the bone screw breaks, and the maximum stress (in N) is recorded. The shear mechanical test is carried out in dry and wet states respectively, and the wet state is prepared by soaking the bone screws in PBS solution at 37℃ for more than 72 hours.

[0496] The results are shown in Figure 21. The experiment shows that the double shear strength of the RSF / G bone screw, RSF bone screw and PLGA bone screw is 259N, 222N and 162N respectively in dry state; and the double shear strength of the RSF / G bone screw, RSF bone screw and PLGA bone screw is 191N, 186N and 163N respectively in wet state. It can be seen that the double shear strength of the RSF / G bone screw is higher than that of the RSF bone screw and the PLGA bone screw, whether in dry state or in wet state.

[0497] The above experimental results show that the shear mechanical strength of the regenerated silk fibroin / gelatin composite bone screw of the application is higher than that of the RSF bone screw and the PLGA bone screw, and the pull-out force is generally equivalent to that of the RSF bone screw and the PLGA bone screw, which has a good clinical application prospect.

[0498] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, all possible combinations of the technical features in the above-described embodiments are not described, but as long as the combinations of the technical features do not contradict, they should be considered within the scope of the present application.

[0499] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are within the scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing a regenerated silk fibroin material, characterized by, The application relates to a regenerated silk fibroin material and a preparation method thereof. The solution containing regenerated silk fibroin is subjected to gel treatment to obtain the regenerated silk fibroin material. The solution containing regenerated silk fibroin comprises a regenerated silk fibroin solution and a regenerated silk fibroin / gelatin composite solution obtained by mixing the regenerated silk fibroin solution with a gelatin solution. The regenerated silk fibroin material comprises a regenerated silk fibroin hard material, a regenerated silk fibroin / gelatin hard material and a regenerated silk fibroin / gelatin composite material.

2. The production method according to claim 1, characterized by, The preparation method of the regenerated silk fibroin solution comprises the following steps: The cocoon is cut into pieces, put into a soap salt solution for degumming treatment, repeatedly washed by pure water or deionized water, and dried to obtain degummed silk fibers, i.e. silk fibroin fibers, wherein the soap salt is sodium carbonate or sodium bicarbonate; The silk fibroin fibers are put into a lithium bromide solution for dissolution, and filtered to obtain a silk fibroin salt solution; The silk fibroin salt solution is subjected to dialysis and desalination to obtain the regenerated silk fibroin solution; The regenerated silk fibroin solution is obtained by reverse dialysis and concentration of the silk fibroin solution by using polyethylene glycol; and / or The preparation method of the regenerated silk fibroin / gelatin composite solution comprises the following steps: The regenerated silk fibroin solution is added with horseradish peroxide and hydrogen peroxide solution respectively to obtain a regenerated silk fibroin composite solution; The regenerated silk fibroin composite solution is subjected to mechanical shaking to make the regenerated silk fibroin composite solution uniformly mixed; or The regenerated silk fibroin solution is mixed with a gelatin solution, and hydrogen peroxide solution, transglutaminase and horseradish peroxide are added in sequence to obtain the regenerated silk fibroin / gelatin composite solution; The regenerated silk fibroin / gelatin composite solution is subjected to mechanical shaking to make the regenerated silk fibroin / gelatin composite solution uniformly mixed; and / or The gel treatment comprises the following steps: The regenerated silk fibroin solution is subjected to water bath gel to obtain a gel block, and the gel block is dried; or The regenerated silk fibroin solution is subjected to water bath gel to obtain a gel block; The gel block is soaked in a methanol solution or an ethanol solution to induce crystallization of the gel block, and a regenerated silk fibroin hard gel block is obtained; The regenerated silk fibroin hard gel block is taken out from the methanol solution or the ethanol solution, and dried to obtain a regenerated silk fibroin hard material; or The regenerated silk fibroin solution is subjected to water bath gel to obtain a gel block; The regenerated silk fibroin / gelatin composite solution is subjected to water bath gel to obtain a gel block, and the gel block is dried; or The regenerated silk fibroin / gelatin composite solution is subjected to water bath gel to obtain a gel block; The gel block is soaked in a methanol solution or an ethanol solution to induce crystallization of the gel block, and a regenerated silk fibroin / gelatin hard gel block is obtained; The regenerated silk fibroin / gelatin hard gel block is taken out from the methanol solution or the ethanol solution, and dried to obtain a regenerated silk fibroin / gelatin hard material; or The regenerated silk fibroin / gelatin composite solution is subjected to water bath gel to obtain a gel block; The gel block is soaked in a methanol solution or an ethanol solution to induce crystallization of the gel block, and a regenerated silk fibroin / gelatin hard gel block is obtained; The regenerated silk fibroin / gelatin hard gel block is soaked in an amine sulfate solution for immersion and cleaning to obtain a regenerated silk fibroin / gelatin crosslinked gel; Drying the regenerated silk fibroin / gelatin crosslinked gel to obtain a regenerated silk fibroin / gelatin hard material.

3. The preparation method according to claim 2, characterized in that, The concentration of the soap salt solution is 0.01M-0.05M; and / or The concentration of the lithium bromide solution is 8-10M; and / or The molecular weight of the polyethylene glycol is 10000Da-30000Da, and the concentration of the polyethylene glycol is 10%-20%; and / or The concentration of the regenerated silk fibroin solution is 5%-20%; and / or The silk is repeatedly washed with deionized water, and dried to obtain a silk fiber, wherein the drying temperature is 35°C-45°C, and the drying time is 20h-30h; and / or The fibroin stock solution is dialyzed and desalted to obtain a fibroin solution, wherein the dialysis molecular weight is 8000Da-14000Da, the dialysis temperature is 20°C-30°C, and the dialysis time is 48h-96h; and / or The fibroin stock solution is replaced with 4-6 times of aqueous solution during dialysis; and / or The concentration of the hydrogen peroxide aqueous solution is 0.1%-1.0%, and the dosage of the hydrogen peroxide aqueous solution is 500-1000μl; and / or The dosage of the transglutaminase is 10-200μl, and the enzyme activity of the transglutaminase is 10-100U / μl; and / or The dosage of the horseradish peroxidase is 500-1000μl, and the enzyme activity of the horseradish peroxidase is 600-1000U / mL; and / or The size of the fibroin / gelatin gel block is 2-4mm; and / or The concentration of the ammonium sulfate solution is 10%-20%, and the dosage of the ammonium sulfate solution is 100-200ml; and / or The drying time is 2-8 days; And / or The fibroin / gel composite solution is horizontally flowed into a mold to gel, to obtain a fibroin / gelatin gel block, which comprises: The fibroin / gel composite solution is mechanically shaken to mix the fibroin / gel composite solution uniformly and form bubbles uniformly distributed in the fibroin / gel composite solution, and the uniformly mixed fibroin / gel composite solution is horizontally flowed into a mold to gel; After the preliminary gelation, the gel is cut to obtain a plurality of fibroin / gelatin gel blocks.

4. The production method according to any one of claims 1 to 3, characterized by, Further comprising: The regenerated silk fibroin-containing solution is subjected to bubble removal treatment to obtain a high-purity regenerated silk fibroin solution or a regenerated silk fibroin / gel composite solution; The high-purity regenerated silk fibroin solution or the regenerated silk fibroin / gel composite solution is subjected to gel treatment to obtain a regenerated silk fibroin gel block or a regenerated silk fibroin / gel hard gel block.

5. The production method according to claim 4, characterized by, The bubble removal treatment comprises: The regenerated silk fibroin composite solution is placed in a centrifuge tube; The centrifuge tube is placed in a centrifuge for centrifugal treatment to remove bubbles in the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained; or The regenerated silk fibroin composite solution is horizontally flowed into a mold; The mold is placed in an ultrasonic machine for ultrasonic treatment to remove bubbles in the regenerated silk fibroin composite solution, and a regenerated silk fibroin / gel composite solution is obtained; or The regenerated silk fibroin composite solution is horizontally flowed into a mold; placing a weight on the panel of the mold to extrude the regenerated silk fibroin composite solution in the mold to remove the bubbles inside the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin / gel composite solution; or directly gelling the regenerated silk fibroin composite solution in the extrusion mold; placing the mold in a vacuum environment to remove the bubbles inside the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin / gel composite solution; or advection of the regenerated silk fibroin composite solution into the mold; placing the mold in a low-temperature environment to freeze the regenerated silk fibroin composite solution, and obtaining a composite solution frozen block; dissolving the composite solution frozen block, and obtaining a regenerated silk fibroin composite solution; repeating the above steps 1-2 times to remove the bubbles inside the regenerated silk fibroin composite solution, and obtaining a regenerated silk fibroin / gel composite solution.

6. The production method according to claim 5, wherein the centrifugal speed for processing the regenerated silk fibroin composite solution is 6000-10000 rpm, and the centrifugal time is 5-60 min; and / or the ultrasonic frequency for processing the regenerated silk fibroin gel solution is 30-100 Hz; and / or the extrusion time for processing the regenerated silk fibroin composite solution is 30-180 min; and / or the vacuum degree for processing the regenerated silk fibroin composite solution is 0.1-0.3 MPa, and the vacuum time is 30-180 min; and / or the freezing temperature for processing the regenerated silk fibroin composite solution is -50--80℃, and the freezing time is 3-7 days; and / or the water bath temperature is 30℃-40℃, and the water bath time is 6h-24h; and / or the soaking time is 24h-36h; and / or the drying time is 4-7 days.

7. The preparation method according to claim 1, characterized in that, comprising: cutting the cocoon, boiling in a 0.02M concentration of sodium carbonate-sodium bicarbonate buffer solution for degumming treatment, and obtaining silk; repeatedly washing the silk with deionized water, and drying the silk fiber at 40℃ for 24h to obtain the silk fiber; dissolving the silk fiber in a 9M concentration of lithium bromide solution, and filtering to obtain a silk fibroin stock solution; dialysis and desalination of the silk fibroin stock solution under the condition of 10000 Da to obtain a silk fibroin solution; after reverse dialysis and concentration of the silk fibroin solution using a 15% concentration and 20000 Da molecular weight polyethylene glycol, a 10% concentration regenerated silk fibroin solution is obtained; adding 700μl of horseradish peroxidase with an enzyme activity of 500U / mL and 700μl of 0.5% concentration hydrogen peroxide aqueous solution to 20ml of the regenerated silk fibroin solution, and mixing uniformly to obtain a silk fibroin composite solution; or mixing the 10% concentration regenerated silk fibroin solution with a 30% concentration gelatin solution, and sequentially adding 700μl of 0.5% concentration hydrogen peroxide aqueous solution, 100μl of 50U / μl transglutaminase, and 700μl of 500U / mL horseradish peroxidase, and mixing uniformly to obtain a regenerated silk fibroin / gel composite solution; The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or The regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution is placed in a centrifuge tube and placed in a centrifuge for centrifugal treatment at a centrifugal speed of 8000 rpm and a centrifugal time of 30 min to remove the bubbles inside the regenerated silk fibroin composite solution or the regenerated silk fibroin / gelatin composite solution, and obtain a regenerated silk fibroin / gel composite solution or a regenerated silk fibroin / gelatin gel composite solution; or placing the regenerated silk fibroin gel block or the regenerated silk fibroin / gelatin gel block in a methanol solution for 30 hours to induce crystallization of the gel block, thereby obtaining a regenerated silk fibroin hard gel block or a regenerated silk fibroin / gelatin hard gel block; or placing the regenerated silk fibroin gel block or the regenerated silk fibroin / gelatin gel block in a methanol solution for 30 hours to induce crystallization of the gel block, thereby obtaining a regenerated silk fibroin hard gel block or a regenerated silk fibroin / gelatin hard gel block; removing the regenerated silk fibroin hard gel block or the regenerated silk fibroin / gelatin hard gel block from the methanol solution or the ethanol solution and drying, thereby obtaining a regenerated silk fibroin hard material or a regenerated silk fibroin / gelatin hard material; or immersing the regenerated silk fibroin / gelatin hard gel block in a 15% ammonium sulfate solution to obtain a regenerated silk fibroin / gelatin crosslinked gel; drying the regenerated silk fibroin hard gel block to obtain a regenerated silk fibroin hard material; or drying the regenerated silk fibroin / gelatin hard gel block or the regenerated silk fibroin / gelatin crosslinked gel to obtain a regenerated silk fibroin / gelatin hard material; or cutting the cocoon, boiling and degumming the cocoon in a 0.02M sodium carbonate-sodium bicarbonate buffer solution, and obtaining silk; washing the silk repeatedly with deionized water and drying the silk at 40°C for 24 hours to obtain silk fibers; dissolving the silk fibers in a 9M lithium bromide solution and filtering to obtain a silk fibroin stock solution; dialyzing and desalting the silk fibroin stock solution under a 10,000 Da condition to obtain a silk fibroin solution; concentrating the silk fibroin solution by reverse dialysis using a 15% polyethylene glycol with a molecular weight of 20,000 Da to obtain a 10% regenerated silk fibroin solution; mixing the 10% regenerated silk fibroin solution with a 30% gelatin solution, and sequentially adding a 0.5% hydrogen peroxide aqueous solution with a dosage of 700 μl, a transglutaminase with an enzyme activity of 50 U / μl and a dosage of 100 μl, and a horseradish peroxidase with an enzyme activity of 500 U / mL and a dosage of 700 μl, and obtaining a gel solution; mechanically shaking the gel solution to uniformly mix the gel solution and form uniformly distributed bubbles in the gel solution, and horizontally flowing the uniformly mixed gelatin solution into a mold to form a gel; cutting the gel after preliminary gelation to obtain a plurality of regenerated silk fibroin / gelatin gel blocks with a size of 3 mm; immersing and washing the regenerated silk fibroin / gelatin gel block in a 15% ammonium sulfate solution to obtain a regenerated silk fibroin / gelatin crosslinked gel; freeze-drying the regenerated silk fibroin / gelatin crosslinked gel for 5 days to obtain a regenerated silk fibroin / gelatin composite bone repair material.

8. A hard material containing regenerated silk fibroin, characterized by, The hard material includes a regenerated silk fibroin hard material and a regenerated silk fibroin / gelatin hard material; The hard material is prepared by the preparation method of any one of claims 1-7.

9. An internal fixation system, characterized in that The hard material includes a bone screw and / or a bone plate; The bone screw and / or the bone plate are prepared from the hard material of claim 8. The hard material includes a bone screw and / or a bone plate. The bone nail and / or bone plate are processed by the following method: a hard material is molded into a columnar or block-shaped parent embryo, and is processed by a lathe to obtain the bone nail and / or bone plate. The structural strength of the bone nail and / or bone plate is 15-500 MPa in a dry state and 5-50 MPa in a wet state.

10. Use of a hard material containing regenerated silk fibroin in the preparation of an internal fixation system for use in trauma orthopedics, plastic surgery (craniofacial), characterized in that, The hard material is prepared by the preparation method of any one of claims 1-7 or is the hard material of claim 8 or the internal fixation system of claim 9.

11. A regenerated silk fibroin / gelatin composite material, characterized by, Prepared by the preparation method of any one of claims 1-7.

12. The regenerated silk fibroin / gelatin composite material according to claim 11, characterized by, The regenerated silk fibroin / gelatin composite material is arranged in a regular particle, an irregular particle, a spherical solid, a cylindrical solid, a cubic solid, a rectangular solid, or an irregularly shaped solid. The regenerated silk fibroin / gelatin composite material is arranged in a porous structure in a micron or nanometer scale, and the porosity of the regenerated silk fibroin / gelatin composite material is 20%-90%. The structural strength (compressive strength) of the regenerated silk fibroin / gelatin composite material is 0.1-35 MPa.

13. The use of regenerated silk fibroin / gelatin composite material in the preparation of bone filling material, which is applied to the filling and regenerative repair of bone defects of neurosurgery skull, orthopedics, oral, plastic surgery (maxillofacial), characterized in that, The regenerated silk fibroin / gelatin composite material is prepared by the preparation method of any one of claims 1-7 or is the regenerated silk fibroin / gelatin composite material of any one of claims 11-12.

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