A high-strength silk fibroin / hydroxyapatite bone repair material and its preparation method

Abstract

This invention discloses a high-strength silk fibroin / hydroxyapatite bone repair material and its preparation method, belonging to the field of biomedical materials. Methacrylated silk fibroin and methacrylated nano-hydroxyapatite are used as organic and inorganic fillers, respectively. Photocrosslinking improves the interfacial bonding and dispersion stability between the organic component silk fibroin and the inorganic component hydroxyapatite. The silk fibroin / hydroxyapatite bone repair material is obtained after high-temperature hot pressing. This bone repair material possesses strong mechanical properties, osteogenic induction ability, good biocompatibility, and degradability, showing broad application prospects in the field of bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material. Background Technology

[0002] Bone tissue possesses self-repairing capabilities and can effectively heal small injuries. However, bone defects exceeding a critical size are difficult to heal spontaneously, such as those caused by traumatic injuries, degenerative diseases, congenital defects, or large-area bone defects resulting from surgical tumor removal. To achieve bone regeneration and functional restoration, clinical interventions are typically required, such as autologous bone grafting, allogeneic bone grafting, or fixation using bio-inert metal devices. However, these treatment options all have limitations. Autologous bone grafting is limited by donor scarcity, allogeneic bone grafts carry the risk of disease transmission from the donor, and metal fixation devices require surgical removal later in the treatment process, potentially causing secondary damage. To overcome these limitations, bone tissue engineering has emerged, aiming to create artificial bone that promotes bone tissue regeneration and repair by utilizing the synergistic effects of bio-scaffold materials, cells, and growth factors. Among these, organic-inorganic composite scaffold materials, as common composite scaffold materials in bone tissue engineering, possess many superior properties.

[0003] Silk fibroin is a natural protein extracted from silkworm silk, possessing excellent biocompatibility and diverse physicochemical properties. Due to its structural similarity to type I collagen, silk fibroin is considered a preferred natural polymer material for bone tissue engineering. However, bone repair materials made from pure silk fibroin exhibit poor mechanical and osteogenic properties. To improve this, hydroxyapatite is added to silk fibroin as a matrix to mimic the natural structure and properties of bone. Chinese invention patent CN112972766B discloses a silk fibroin-hydroxyapatite composite bone scaffold and its preparation method. A silk fibroin solution and a hydroxyapatite aqueous dispersion are mixed by ultrasonic treatment, and a three-dimensional porous silk fibroin / hydroxyapatite composite scaffold is rapidly and non-toxically prepared using a DC directional electric field induction method. However, the silk fibroin / hydroxyapatite composite system obtained by simple physical mixing lacks a strong interfacial bond between the organic components of silk fibroin and the inorganic components of hydroxyapatite. This leads to the agglomeration of hydroxyapatite within the system, resulting in uneven dispersion and affecting the mechanical properties and osteoinductive activity of the scaffold. Chinese invention patent CN110522950A discloses a silk fibroin / hydroxyapatite composite scaffold and its preparation method. Microwave-assisted mineralization of a silk fibroin scaffold immersed in SBF solution is used to deposit hydroxyapatite on the surface and interior of the scaffold, thus obtaining the silk fibroin / hydroxyapatite composite scaffold. While the aforementioned patent uses substrate mineralization deposition to obtain the silk fibroin / hydroxyapatite composite scaffold, which improves the interfacial bond between the organic components of silk fibroin and the inorganic components of hydroxyapatite, the hydroxyapatite content is limited, affecting the full integration of the organic and inorganic components within the scaffold. This results in low mechanical properties and weak osteoinductive activity, presenting certain defects when used as a bone repair material. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material. This method introduces covalent bonds by methacrylate esterification of silk fibroin and nano-hydroxyapatite, thereby improving the interfacial bonding ability and dispersion stability between the organic components of silk fibroin and the inorganic components of nano-hydroxyapatite. High-temperature hot pressing molding further enhances the mechanical properties and osteoinductive activity of the bone repair material, solving the problem of unstable mechanical properties and poor bioactivity in traditional organic-inorganic bone tissue engineering composite scaffold materials due to weak interfacial bonding of internal components.

[0005] The present invention adopts the following technical solution:

[0006] A method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material: methacrylated silk fibroin and methacrylated nano-hydroxyapatite are mixed, and the two are covalently cross-linked under the action of a photoinitiator. Then, the mixture is subjected to high-temperature hot pressing to obtain the high-strength silk fibroin / hydroxyapatite bone repair material.

[0007] The preparation method of the above-mentioned high-strength silk fibroin / hydroxyapatite bone repair material specifically includes the following steps:

[0008] (1) Preparation of methacrylated silk fibroin solution: Deionized water was heated to boiling, and anhydrous sodium carbonate and silk were added to degumm the silk. After washing and drying, the dried degummed silk was dissolved in lithium bromide solution to obtain a silk fibroin / lithium bromide mixed solution. Then, glycidyl methacrylate was added to modify the silk fibroin in the dark for 3 hours. Then, the solution was dialyzed with deionized water at room temperature in the dark for 5 days. The dialyzed solution was centrifuged and concentrated to obtain a methacrylated silk fibroin solution. The mass fraction of the methacrylated silk fibroin solution was 15%-30%.

[0009] (2) Preparation of methacrylated nano-hydroxyapatite: The dehydrated nano-hydroxyapatite and isophorone diisocyanate were heated and stirred in an oil bath, followed by the addition of dibutyltin dilaurate and N,N-dimethylformamide, and the reaction was carried out in a nitrogen atmosphere. Then, 2-hydroxyethyl methacrylate was added and the stirring was continued. Finally, methanol was added to terminate the reaction. The precipitate was collected by centrifugation, washed with dichloromethane, dried at room temperature, and ground to obtain methacrylated nano-hydroxyapatite powder.

[0010] (3) Preparation of silk fibroin / hydroxyapatite crosslinking system: The methacrylated silk fibroin solution obtained in step (1) is mixed with a photoinitiator, and then the methacrylated nano-hydroxyapatite powder obtained in step (2) is added. The mixture is stirred and mixed evenly at room temperature in the dark to obtain a precursor slurry of bone repair material; the precursor slurry is irradiated under ultraviolet light to obtain the silk fibroin / hydroxyapatite crosslinking system.

[0011] (4) Preparation of high-strength silk fibroin / hydroxyapatite bone repair material: The silk fibroin / hydroxyapatite crosslinking system prepared in step (3) was vacuum freeze-dried at -80℃, and then crushed and ground into powder using a high-speed crusher at a speed of 39000 r / min. The powder was hot-pressed for 20 minutes under a pressure of 2 MPa and a temperature of 200℃ to obtain high-strength silk fibroin / hydroxyapatite bone repair material.

[0012] Furthermore, in step (2), the reaction temperature for adding 2-hydroxyethyl methacrylate and continuing stirring is 40-80 °C, the reaction time is 1-5 h, and the reaction is carried out in the dark.

[0013] Furthermore, the mass fraction of the methacrylated nano-hydroxyapatite powder in step (3) in the precursor slurry of the bone repair material is 10wt%-50wt%.

[0014] Further, the photoinitiator mentioned in step (3) is one of Irgacure 2959, Ruthenium Photoinitiator and phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite.

[0015] Furthermore, the wavelength of the ultraviolet light in step (3) is 365 nm, and the irradiation time is 5-20 min.

[0016] A high-strength silk fibroin / hydroxyapatite bone repair material prepared by the above-mentioned preparation method.

[0017] The application of the aforementioned high-strength silk fibroin / hydroxyapatite bone repair material to drug loading involves adding the drug to the precursor slurry of the bone repair material.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) This invention involves using a photoinitiator to induce a chemical cross-linking reaction between methacrylated silk fibroin and methacrylated nano-hydroxyapatite, forming stable covalent bonds and improving the interfacial bonding between organic and inorganic materials and the dispersibility of nano-hydroxyapatite in bone repair materials. Compared with traditional organic-inorganic composite scaffolds, this invention solves the problems of weak interfacial bonding between organic and inorganic materials, uneven distribution of nano-hydroxyapatite in bone repair materials, insufficient mechanical properties, and weak osteoinductive activity. It has significantly enhanced mechanical properties, good biocompatibility, and degradability, providing a solution for bone defect repair.

[0020] (2) This invention successfully prepared methacrylated nano-hydroxyapatite using a two-step method. First, isophorone diisocyanate reacts with the hydroxyl groups on the surface of nano-hydroxyapatite, fixing them onto the surface and providing active sites. Then, these sites react with 2-hydroxyethyl methacrylate, introducing methacrylate groups and enhancing the content and chemical activity of methacrylate groups on the surface of nano-hydroxyapatite. This allows it to bind more fully with methacrylated modified silk fibroin, forming a composite bone repair material with higher density and better mechanical properties under the action of a photoinitiator.

[0021] (3) The high-strength silk fibroin / hydroxyapatite bone repair material of the present invention is formed by hot pressing covalently cross-linked silk fibroin / hydroxyapatite composite powder under a pressure of 2 MPa and a temperature of 200°C for 20 minutes, which is conducive to the interface fusion between the organic component silk fibroin and the inorganic component hydroxyapatite in the composite material, forming a high-strength silk fibroin / hydroxyapatite bone repair material with a uniform and complete interface.

[0022] (4) The high-strength silk fibroin / hydroxyapatite bone repair material of the present invention can be loaded with different types of drugs, such as bone morphogenetic protein-2, to improve the bioactivity of the scaffold and significantly enhance the osteogenic induction ability, so that the scaffold can act as a drug carrier to achieve drug sustained release effect, and become an ideal bone repair scaffold material.

[0023] (5) The high-strength silk fibroin / hydroxyapatite bone repair material of the present invention has good biocompatibility and biodegradability, low cost, simple operation and easy mass production. Attached Figure Description

[0024] Figure 1 : 1H NMR spectrum of silk fibroin and methacrylated silk fibroin.

[0025] Figure 2 Infrared spectra of nano-hydroxyapatite, 2-hydroxyethyl methacrylate, and methacrylate-esterified nano-hydroxyapatite.

[0026] Figure 3 : Interfacial binding diagram of methacrylated silk fibroin with (A) nano-hydroxyapatite and (B) methacrylated nano-hydroxyapatite under a scanning electron microscope.

[0027] Figure 4 Comparison of the compressive modulus of silk fibroin / hydroxyapatite bone repair materials prepared with different contents of nano-hydroxyapatite or methacrylated nano-hydroxyapatite.

[0028] Figure 5 Release curves of BMP-2 in silk fibroin / hydroxyapatite bone repair material under different pH conditions. Detailed Implementation

[0029] To verify the feasibility of the design, the technical solution of the present invention will be further described below with reference to specific embodiments, but the application of the present invention is not limited thereto.

[0030] Example 1

[0031] (1) Preparation of methacrylated silk fibroin solution: Boil 3 L of deionized water and add 15.9 g of anhydrous sodium carbonate. After it is fully dissolved, weigh 60 g of silk and put it into the solution. Degumming treatment at 100 ℃ for 30 min. Take out the boiled silk and wash it thoroughly with deionized water. Dry it in an oven at 60 ℃ to obtain degummed silk. Add 40 g of dried degummed silk to 200 mL of 9.3 M LiBr solution and dissolve it at 60 ℃ for 1 h to obtain a silk fibroin / lithium bromide mixed solution. Then add 12 mL of glycidyl methacrylate and stir at 60 ℃ in the dark for 3 h. After the reaction is completed, transfer the solution to a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyze it with deionized water for 5 days. The dialysis process is carried out in the dark. After dialysis, the solution is centrifuged and concentrated to obtain a methacrylated silk fibroin solution with a mass fraction of about 25%.

[0032] (2) Preparation of methacrylated nano-hydroxyapatite: 10.5 g of nano-hydroxyapatite was dried in an oven at 120 °C to remove moisture. 10 g of dried nano-hydroxyapatite and 5 mL of isophorone diisocyanate were added to a three-necked flask and sealed. The flask was heated and stirred in an oil bath at 50 °C. 0.5 mL of dibutyltin dilaurate and 300 mL of N,N-dimethylformamide were added to the flask and nitrogen gas was introduced. After reacting for 48 h, 6 mL of 2-hydroxyethyl methacrylate was added and stirring was continued for 8 h. Then, 100 mL of methanol was added to terminate the reaction. The precipitate was collected by centrifugation, washed with dichloromethane, dried at room temperature for 48 h, and then ground into powder to obtain methacrylated nano-hydroxyapatite.

[0033] (3) Preparation of silk fibroin / hydroxyapatite crosslinking system: Add 0.3% phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite (LAP) to 1 mL of methacrylated silk fibroin solution with a mass fraction of 25%, and then add 10wt% methacrylated nano-hydroxyapatite powder. Stir and mix evenly at room temperature in the dark to obtain a precursor slurry of bone repair material; irradiate the precursor slurry under 365 nm ultraviolet light for 10 min to obtain the silk fibroin / hydroxyapatite crosslinking system.

[0034] (4) Preparation of high-strength silk fibroin / hydroxyapatite bone repair material: The prepared silk fibroin / hydroxyapatite crosslinking system was vacuum freeze-dried at -80℃ for 24 hours, and then crushed and ground into powder using a high-speed crusher at 39000 r / min for 30 minutes. The powder was hot-pressed at a pressure of 2 MPa and a temperature of 200℃ for 20 minutes to obtain high-strength silk fibroin / hydroxyapatite bone repair material.

[0035] like Figure 1 As shown, the methacrylated silk fibroin prepared in this embodiment has characteristic peaks of vinyl methacrylate (δ=6.2-6 ppm and 5.8-5.6 ppm) and methyl methacrylate (δ=1.8 ppm) in the glycidyl methacrylate molecule, which proves that glycidyl methacrylate chemically modifies silk fibroin and successfully prepares methacrylated modified silk fibroin.

[0036] like Figure 2 As shown in the infrared spectrum, the methacrylated nano-hydroxyapatite prepared in this embodiment exhibits the characteristic peak of 2-hydroxyethyl methacrylate (2940 cm⁻¹). -1 -CH2 group at 1640 cm -1 The presence of C=C groups at the methyl methacrylate (MCM) indicates the successful synthesis of nano-hydroxyapatite.

[0037] Example 2

[0038] Except that the mass fraction of methacrylated nano-hydroxyapatite powder added in step (3) is changed to 30wt%, the other steps are the same as in Example 1.

[0039] Example 3

[0040] Except that the mass fraction of methacrylated nano-hydroxyapatite powder added in step (3) is changed to 50wt%, the other steps are the same as in Example 1.

[0041] Comparative Example 1

[0042] Except for the addition of unmethacrylated nano-hydroxyapatite powder in step (3), the other steps are the same as in Example 1.

[0043] Comparative Example 2

[0044] Except for the addition of unmethacrylated nano-hydroxyapatite powder in step (3), the other steps are the same as in Example 2.

[0045] Comparative Example 3

[0046] Except for the addition of unmethacrylated nano-hydroxyapatite powder in step (3), the other steps are the same as in Example 3.

[0047] like Figure 3 As shown, from Figure 3(A) It can be seen that in the bone repair material prepared using unmodified nano-hydroxyapatite in Comparative Example 1, hydroxyapatite agglomerated extensively in the silk fibroin network and was not uniformly dispersed. In contrast, as shown in Figure 3 (B), in the bone repair material prepared using methacrylate-modified nano-hydroxyapatite in Example 1, hydroxyapatite did not aggregate but was uniformly dispersed in the silk fibroin network and tightly adhered to the inner walls of the pores of the silk fibroin scaffold, demonstrating a good binding and dispersion effect between silk fibroin and hydroxyapatite.

[0048] like Figure 4 As shown, a comparison of compressive moduli reveals that, under the same inorganic content, the compressive modulus of the methacrylate-modified nano-hydroxyapatite group is significantly greater than that of the unmodified nano-hydroxyapatite group. The compressive modulus of the silk fibroin / hydroxyapatite bone repair material prepared in Comparative Example 3 is 17.08 MPa, significantly lower than the compressive modulus of the silk fibroin / hydroxyapatite bone repair material prepared in Example 3 (46.33 MPa). These results indicate that, compared to unmodified nano-hydroxyapatite, the bone repair material prepared using methacrylate-modified nano-hydroxyapatite significantly improves the mechanical properties of the bone repair material.

[0049] Comparative Example 4

[0050] Steps (1) and (2) are the same as in Example 3;

[0051] (3) Preparation of silk fibroin / hydroxyapatite bone repair material: Add 0.3% phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite (LAP) to 1 mL of 25% methacrylated silk fibroin solution, and then add 50 wt% methacrylated nano-hydroxyapatite powder. Stir and mix evenly at room temperature in the dark to obtain the precursor slurry of bone repair material; irradiate the precursor slurry under 365 nm ultraviolet light for 10 min to obtain silk fibroin / hydroxyapatite bone repair material.

[0052] The compressive modulus of the silk fibroin / hydroxyapatite bone repair material prepared in Comparative Example 4 using the above steps was 12.69 MPa, significantly lower than the compressive modulus (46.33 MPa) of the silk fibroin / hydroxyapatite bone repair material prepared in Example 3. This result indicates that in Example 3, the powder prepared by vacuum freeze-drying and grinding the covalently cross-linked silk fibroin / hydroxyapatite cross-linking system at -80°C, followed by hot pressing at 2 MPa and 200°C for 20 minutes, facilitates the interfacial fusion between the organic component silk fibroin and the inorganic component hydroxyapatite within the composite material. This results in a high-strength silk fibroin / hydroxyapatite bone repair material with a uniform and complete interface, thereby significantly improving the mechanical properties of the bone repair material.

[0053] Application Example 1

[0054] Steps (1) and (2) are the same as in Example 1;

[0055] (3) Preparation of silk fibroin / hydroxyapatite crosslinking system: Add 0.3% phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite (LAP) to 1 mL of 25% methacrylated silk fibroin solution, then add 10 wt% methacrylated nano-hydroxyapatite powder. Stir and mix evenly at room temperature in the dark to obtain a precursor slurry for bone repair material. Add 3 μg / mL bone morphogenetic protein-2 (BMP-2) drug to it and stir and mix evenly. Finally, irradiate the obtained precursor slurry containing BMP-2 drug under 365 nm ultraviolet light for 10 min to obtain a silk fibroin / hydroxyapatite crosslinking system loaded with BMP-2 drug.

[0056] (4) Preparation of high-strength silk fibroin / hydroxyapatite bone repair material: The prepared silk fibroin / hydroxyapatite crosslinking system was vacuum freeze-dried at -80℃ for 24 hours, and then crushed and ground into powder using a high-speed crusher at 39000 r / min for 30 minutes. The powder was hot-pressed at a pressure of 2 MPa and a temperature of 200℃ for 20 minutes to obtain high-strength silk fibroin / hydroxyapatite bone repair material.

[0057] like Figure 5 As shown, the high-strength silk fibroin / hydroxyapatite bone repair material loaded with BMP-2 can slowly release BMP-2. These results confirm that the high-strength silk fibroin / hydroxyapatite bone repair material can serve as a drug sustained-release carrier, continuously releasing the loaded drug and enhancing the material's bioactivity.

[0058] The above description is merely an embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention shall be covered by the present invention.

Claims

1. A method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material, characterized in that: Specifically, the following steps are included: (1) Preparation of methacrylated silk fibroin solution: After heating deionized water to boiling, anhydrous sodium carbonate and silk were added to degumm the silk; after washing and drying, the dried degummed silk was dissolved in lithium bromide solution to obtain a silk fibroin / lithium bromide mixed solution. Then, glycidyl methacrylate was added to modify the silk fibroin in the dark for 3 hours. Then, the solution was dialyzed with deionized water at room temperature in the dark for 5 days. The dialyzed solution was centrifuged and concentrated to obtain a methacrylated silk fibroin solution; the mass fraction of the methacrylated silk fibroin solution was 15%-30%. (2) Preparation of methacrylated nano-hydroxyapatite: The dehydrated nano-hydroxyapatite and isophorone diisocyanate were heated and stirred in an oil bath, followed by the addition of dibutyltin dilaurate and N,N-dimethylformamide, and the reaction was carried out in a nitrogen atmosphere. Then, 2-hydroxyethyl methacrylate was added and the stirring was continued. Finally, methanol was added to terminate the reaction. The precipitate was collected by centrifugation, washed with dichloromethane, dried at room temperature, and ground to obtain methacrylated nano-hydroxyapatite powder. (3) Preparation of silk fibroin / hydroxyapatite crosslinking system: The methacrylated silk fibroin solution obtained in step (1) is mixed with a photoinitiator, and then the methacrylated nano-hydroxyapatite powder obtained in step (2) is added. The mixture is stirred and mixed evenly at room temperature in the dark to obtain a precursor slurry of bone repair material; the precursor slurry is irradiated under ultraviolet light to obtain the silk fibroin / hydroxyapatite crosslinking system. (4) Preparation of high-strength silk fibroin / hydroxyapatite bone repair material: The silk fibroin / hydroxyapatite crosslinking system prepared in step (3) was vacuum freeze-dried at -80℃, and then crushed and ground into powder by a high-speed crusher at a speed of 39000 r / min. The powder was hot-pressed for 20 minutes under a pressure of 2 MPa and a temperature of 200℃ to obtain high-strength silk fibroin / hydroxyapatite bone repair material.

2. The method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material according to claim 1, characterized in that: The reaction temperature for adding 2-hydroxyethyl methacrylate and stirring in step (2) is 40-80℃, the reaction time is 1-5 h, and the reaction is carried out in the dark.

3. The method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material according to claim 1, characterized in that: The mass fraction of the methacrylated nano-hydroxyapatite powder in step (3) in the precursor slurry of the bone repair material is 10wt%-50wt%.

4. The method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material according to claim 1, characterized in that: The photoinitiator mentioned in step (3) is one of Irgacure 2959, Ruthenium Photoinitiator and phenyl-2,4,6-trimethylbenzoyl lithium hypophosphite.

5. The method for preparing a high-strength silk fibroin / hydroxyapatite bone repair material according to claim 1, characterized in that: The wavelength of the ultraviolet light in step (3) is 365 nm, and the irradiation time is 5-20 min.

6. A high-strength silk fibroin / hydroxyapatite bone repair material prepared by the preparation method according to any one of claims 1 to 5.

7. The application of the high-strength silk fibroin / hydroxyapatite bone repair material as described in claim 6 on drug loading, characterized in that: The drug is added to the precursor slurry of the bone repair material.

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