Biomimetic mineralized silk fibroin scaffold material and preparation method and application thereof

Abstract

The present application relates to the field of biomimetic mineralization and tissue engineering scaffold materials, and particularly relates to a kind of biomimetic mineralization silk fibroin scaffold material and its preparation method and application.The preparation method comprises the following steps:1) silk fibroin is induced self-assembly, and silk fibroin fiber matrix is obtained;2) the silk fibroin fiber obtained in step 1) is crosslinked, and crosslinked silk fibroin fiber mineralization matrix is obtained;3) the crosslinked silk fibroin fiber mineralization matrix obtained in step 2) is mineralized in mineralization solution, and biomimetic mineralization silk fibroin scaffold material is obtained.The biomimetic mineralization silk fibroin scaffold material prepared by the preparation method is applied to bone tissue repair.In the present application, the mineralization mode in silk fibroin fiber more highly simulates the mineralization form of natural bone tissue, and the similar surface chemical properties and nano-scale structure characteristics of natural bone are reproduced.

Description

Technical Field

[0001] This invention relates to the field of biomimetic mineralization and tissue engineering scaffold materials, specifically to a biomimetic mineralized silk fibroin scaffold material, its preparation method, and its application. Background Technology

[0002] In today's society, bone defects caused by trauma, tumors, surgery, and other reasons are constantly increasing, leading to a growing demand for bone graft materials. Commonly used bone graft materials in clinical practice include autologous bone, allogeneic bone, and xenogeneic bone, with autologous bone long considered the "gold standard" for bone defect quality. However, these bone graft materials generally suffer from limited availability, complex surgical procedures, immune rejection, and complications. Therefore, the development of highly biomimetic artificial bone materials as alternatives to bone graft donors is crucial to addressing this demand.

[0003] Silk fibroin is a high-performance and readily available natural polymer. Due to its series of small hydrophobic amino acids and large hydrophilic side-chain amino acids, silk fibroin is easily degradable, structurally controllable, and exhibits good bioactivity. With the development of biomaterials, silk fibroin has been widely studied and applied in tissue engineering, tumor therapy, and medical testing. Many methods exist for incorporating hydroxyapatite minerals into silk fibroin, including physically mixing hydroxyapatite of various particle sizes with the silk fibroin matrix; incorporating hydroxyapatite into a silk fibroin solution followed by electrospinning; or depositing hydroxyapatite onto the surface of the silk fibroin matrix via electroplating. However, silk fibroin-hydroxyapatite composites prepared by these methods cannot achieve true biomimicry of natural bone tissue in terms of microstructure and microenvironment, resulting in poor mechanical properties and low bioactivity in these composites.

[0004] Natural bone tissue is a nanocomposite material, its microstructure mainly composed of hydroxyapatite nanocrystals and collagen nanofibers. Through biomineralization, hydroxyapatite mineralized crystals are deposited within collagen fibers, thus endowing natural bone with excellent mechanical properties and maintaining the good bioactivity of collagen. Biomimetic mineralization utilizes the principle of biomineralization to prepare structures similar to natural organic-inorganic composite materials in vitro. Currently, biomimetic mineralization mainly uses collagen fibers as a mineralization matrix to regulate the synthesis of hydroxyapatite, thereby achieving intrafiber mineralization. Studies have shown that the characteristic striation structure (D=67nm) within collagen fibers dominates the regular deposition of hydroxyapatite crystals within the fibers. The intrafiber mineralization scaffold has better mechanical properties and maintains the surface chemical structure of the mineralization matrix, thus maintaining good bioactivity. However, currently, intrafiber mineralization has only been achieved on a limited number of mineralization matrix materials, such as collagen. Collagen supply is limited and expensive; therefore, finding non-collagen mineralization matrices with superior properties to achieve precise biomimetic mineralization is crucial for bone tissue regeneration and repair. Summary of the Invention

[0005] To address the problems existing in the prior art, the first aspect of the present invention provides a biomimetic mineralized silk fibroin scaffold material, the biomimetic mineralized silk fibroin scaffold material comprising a cross-linked silk fibroin fiber mineralization matrix and a mineralization solution.

[0006] A second aspect of the present invention provides a method for preparing the above-mentioned biomimetic mineralized silk fibroin scaffold material, comprising the following steps:

[0007] 1) Silk fibroin was induced to self-assemble in solution to obtain a silk fibroin fiber matrix;

[0008] 2) Crosslink the silk fibroin fiber matrix obtained in step 1) to obtain a crosslinked silk fibroin fiber mineralized matrix;

[0009] 3) The cross-linked silk fibroin fiber mineralized matrix obtained in step 2) is mineralized in a mineralizing solution to obtain the biomimetic mineralized silk fibroin scaffold material.

[0010] Preferably, it includes at least one of the following technical features:

[0011] 11) In step 1), the solution is water;

[0012] 12) In step 1), the temperature for inducing self-assembly is 0-10℃;

[0013] 13) In step 1), the time for inducing self-assembly is ≥1 hour;

[0014] 14) In step 1), the silk fibroin is derived from silkworm silk;

[0015] 15) In step 1), the concentration of the silk fibroin is ≥0.1 mg / mL;

[0016] 21) In step 2), before the crosslinking, the silk fibroin fiber matrix obtained in step 1) is freeze-dried;

[0017] 22) In step 2), the crosslinking is selected from at least one of chemical crosslinking and physical crosslinking;

[0018] 31) In step 3), the mineralizing solution includes calcium salt, stabilizer, and phosphate;

[0019] 32) In step 3), the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralizing matrix is ​​≥1;

[0020] 33) In step 3), the mineralization time is ≥24 hours;

[0021] 34) In step 3), after mineralization, the process also includes washing and freeze-drying.

[0022] Preferably, it includes at least one of the following technical features:

[0023] In feature 12), the temperature for inducing self-assembly is 4-8°C;

[0024] In feature 12), the induced self-assembly time is 1-3 days;

[0025] 151) In feature 15), the concentration of the silk fibroin solution is ≥3 mg / mL, preferably, the concentration of the silk fibroin solution is ≥5 mg / mL.

[0026] In feature 22), when the crosslinking is chemical crosslinking, the silk fibroin fiber matrix is ​​crosslinked with a crosslinking agent;

[0027] 222) In feature 22), when the crosslinking is physical crosslinking, the silk fibroin fiber matrix is ​​dehydrated to obtain a crosslinked silk fibroin fiber mineralized matrix;

[0028] 223) In feature 22), when the crosslinking is physical crosslinking, the temperature of the physical crosslinking is ≥80℃;

[0029] 224) In feature 22), when the crosslinking is physical crosslinking, the pressure of the physical crosslinking is ≥15 psi;

[0030] 225) In feature 22), when the crosslinking is physical crosslinking, the physical crosslinking time is ≥15 min;

[0031] 311) In feature 31), the calcium salt is a water-soluble calcium salt;

[0032] 312) In feature 31), the calcium ion concentration in the mineralizing solution is 0.2-20 mmol / L;

[0033] 313) In feature 31), the stabilizer is selected from one or more of polyaspartic acid, polyaspartic acid salt, polyacrylic acid, and polyacrylate;

[0034] 314) In feature 31), the phosphate is selected from one or more of disodium hydrogen phosphate and dipotassium hydrogen phosphate;

[0035] 315) In feature 31), the concentration of phosphate ions in the mineralizing solution is 0.3-30 mmol / L;

[0036] 321) In feature 32), the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralizing matrix is ​​≥100;

[0037] In feature 33), the mineralization time is ≥3 days;

[0038] In feature 34), the reagent used for cleaning is deionized water.

[0039] Preferably, it includes at least one of the following technical features:

[0040] In feature 221), the crosslinking agent is selected from one or more of glutaraldehyde, carbodiimide, and genipin;

[0041] In feature 311), the calcium salt is selected from one or more of calcium chloride and calcium nitrate;

[0042] In feature 312), the calcium ion concentration is 1-10 mmol / L;

[0043] In feature 313), when the stabilizer is a polyaspartic acid salt, the polyaspartic acid salt is selected from one or more of potassium polyaspartic acid and sodium polyaspartic acid;

[0044] In feature 313), when the stabilizer is polyaspartic acid, the concentration of polyaspartic acid is 10-400 μg / mL; preferably, the concentration of polyaspartic acid is 50-200 μg / mL.

[0045] In feature 313), when the stabilizer is polyaspartic acid, the molecular weight of polyaspartic acid is 3000-50000 g / mol; preferably, the molecular weight of polyaspartic acid is 5000-50000 g / mol; more preferably, the molecular weight of polyaspartic acid is 20000-50000 g / mol.

[0046] In feature 314), the concentration of the phosphate ions is 1-5 mmol / L;

[0047] Preferably, in step 1), the silk fibroin is prepared by degumming, rinsing, drying, dissolving, dialysis, centrifuging and removing impurities from silkworm cocoons to obtain silk fibroin.

[0048] Preferably, it includes at least one of the following technical features:

[0049] A1) The degumming solution is a Na2CO3 solution;

[0050] A2) The degumming method described is boiling degumming;

[0051] A3) The degumming time is 30-60 minutes;

[0052] A4) The rinsing solution is distilled water;

[0053] A5) The drying time is 24-72 hours;

[0054] A6) The solution to be dissolved is a lithium bromide solution;

[0055] A7) The dialysis solution is deionized water;

[0056] A8) The dialysis solution described herein is to be used for 8-12 hours each time;

[0057] A9) The dialysis time is 2-3 days;

[0058] The centrifugation temperature mentioned in A10 is 4-8℃.

[0059] The centrifugation speed described in A11) is 6000-9000 RPM;

[0060] The centrifugation time described in A12) is 5-30 min.

[0061] Preferably, it includes at least one of the following technical features:

[0062] In feature A1), the concentration of the Na2CO3 solution is 0.02-0.1M;

[0063] In feature A2), the boiling degumming temperature is ≥90℃;

[0064] In feature A6), the weight-volume ratio of the dried silk fibroin to the lithium bromide solution is 1:4 to 1:8.

[0065] In feature A6), the concentration of the lithium bromide solution is 9.3-9.5 M.

[0066] Preferably, in step 3), the mineralizing solution is prepared by mixing a first mixture comprising calcium salt, stabilizer and buffer solution and a second mixture comprising phosphate and buffer solution to obtain the mineralizing solution.

[0067] Preferably, it includes at least one of the following technical features:

[0068] B1) The pH of the buffer solution is 7.4-7.8 at 37°C;

[0069] B2) The buffer solution is a Tris buffer solution;

[0070] B3) The buffer solution includes one or more of Trizma HCl, Trizma Base and NaCl.

[0071] A third aspect of the present invention provides an application of a biomimetic mineralized silk fibroin scaffold material in bone tissue repair, wherein the biomimetic mineralized silk fibroin scaffold material is prepared by the method described above.

[0072] The present invention has at least one of the following beneficial effects:

[0073] 1) Unlike traditional methods for preparing silk fibroin-hydroxyapatite composite materials, this invention provides a method for achieving intrafiber mineralization of silk fibroin fibers under conditions closer to physiological realities. The intrafiber mineralization pattern more closely mimics the mineralization form of natural bone tissue, reproducing surface chemical properties and nanoscale structural features similar to natural bone.

[0074] 2) Mineralization within non-collagen fibers was achieved by inducing silk fibroin self-assembly and a biomimetic mineralization process. The intrafiber mineralized silk fibroin scaffold prepared by this method significantly enhances the mechanical strength of silk fibroin fibers and provides an internal environment similar to natural bone for cell growth, thereby promoting the adhesion, proliferation, and osteogenic differentiation of bone marrow mesenchymal stem cells.

[0075] 3) This invention provides a mineralized scaffold material that is easy to prepare, uses readily available raw materials, is inexpensive, and has excellent mechanical and biocompatibility, and is expected to become an ideal material for bone defect regeneration and repair. Attached Figure Description

[0076] Figure 1 The image shows the structural changes of silk fibroin samples after different treatments detected by ATR-FTIR in Example 1. Fresh: Freshly prepared lyophilized silk fibroin solution; 4℃ 1D: Lyophilized silk fibroin sample after induced self-assembly at 4℃ for 1 day; 4℃ 1D+AC: Silk fibroin sample after self-assembly at 4℃ for 1 day, lyophilized, and then cross-linked at high temperature and high pressure for 15 minutes.

[0077] Figure 2 This is a schematic diagram illustrating the analysis of the secondary structure content of silk fibroin using infrared spectroscopy in Example 1. A: Freshly prepared lyophilized silk fibroin solution; B: Lyophilized silk fibroin sample after self-assembly at 4°C for 1 day; C: Silk fibroin sample after self-assembly at 4°C for 1 day, lyophilized, and then cross-linked under high temperature and pressure for 15 minutes. Black solid line: Experimental infrared spectrum; red solid line: Fitted curve; blue solid line: Deconvolution spectrum. RC: Random coil; β-sh: β-sheet; T: β-turn.

[0078] Figure 3This is a schematic diagram of the morphological characteristics of silk fibroin before and after induced self-assembly observed by scanning electron microscopy in Example 1. A: Freshly prepared silk fibroin solution freeze-dried sample; B: Silk fibroin freeze-dried sample after self-assembly at 4°C for 1 day; C: Silk fibroin sample after self-assembly at 4°C for 1 day, freeze-dried, and then cross-linked under high temperature and high pressure for 15 minutes.

[0079] Figure 4 A, C, and D are schematic diagrams of the morphology of the biomimetic mineralized silk fibroin scaffold material after mineralization in Example 1. B: Schematic diagram of EDX elemental analysis of the morphology of the biomimetic mineralized silk fibroin scaffold material after mineralization in Example 1.

[0080] Figure 5 This is a schematic diagram of the ATR-FTIR spectroscopic analysis of the cross-linked silk fibroin fiber mineralized matrix before and after mineralization in Example 1. SF: Unmineralized freeze-dried sample; SF+PILP3D: Biomimetic mineralized silk fibroin scaffold material after 3 days of mineralization.

[0081] Figure 6 This is a scanning electron microscope (SEM) schematic diagram of the silk fibroin matrix that self-assembled at 4°C for 5 days in Example 1, followed by 3 days of biomimetic mineralization. Detailed Implementation

[0082] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0083] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0084] A method for preparing a biomimetic mineralized silk fibroin scaffold material, characterized by comprising the following steps:

[0085] 1) Silk fibroin was induced to self-assemble in solution to obtain a silk fibroin fiber matrix;

[0086] 2) Crosslink the silk fibroin fiber matrix obtained in step 1) to obtain a crosslinked silk fibroin fiber mineralized matrix;

[0087] 3) The cross-linked silk fibroin fiber mineralized matrix obtained in step 2) is mineralized in a mineralizing solution to obtain the biomimetic mineralized silk fibroin scaffold material.

[0088] Specifically, silkworm silk is dissolved to obtain a silk fibroin solution, which is then allowed to stand at low temperature to induce the formation of a silk fibroin fiber matrix. The silk fibroin fiber matrix is ​​freeze-dried and then cross-linked to obtain a cross-linked silk fibroin fiber mineralized matrix. This cross-linked silk fibroin fiber mineralized matrix is ​​then immersed in a mineralizing solution for mineralization, resulting in the biomimetic mineralized silk fibroin scaffold material. This invention induces the self-assembly of silk fibroin to form fibers. Water and amorphous calcium phosphate nanoclusters, stabilized by polyaspartic acid, enter the interior of the silk fibroin fibers, subsequently dehydrating and solidifying to form hydroxyapatite, ultimately forming a mineralized silk fibroin fiber matrix, thus achieving intrafiber mineralization of silk fibroin fibers. The mineralized silk fibroin scaffold material of this invention is highly biomimetic, has high mineralization efficiency, is easy to prepare, has good mechanical properties, and is beneficial for bone tissue regeneration and repair.

[0089] In a preferred embodiment, the silk fibroin is prepared as follows: Dried silkworm cocoons cut into small pieces are boiled in a Na₂CO₃ (0.02-0.1M) aqueous solution for 30-60 minutes to degummify, and then thoroughly rinsed with distilled water. The fibroin is dried in a fume hood for 24-72 hours. The dried fibroin is then added to a lithium bromide solution (9.3-9.5M) at a weight-to-volume ratio and dissolved at 60-70°C for 4-6 hours. The dissolved silk fibroin is purified by dialyzing with 1-2L of deionized water using a dialysis tube (cellulose, MWCO 3kDa) for 2-3 days. The deionized water is changed every 8-12 hours. After dialysis purification, the silk fibroin solution is transferred to a centrifuge tube and centrifuged at 6000-9000 RPM for 5-30 minutes at 4-8°C to remove impurities, thus obtaining the purified silk fibroin solution.

[0090] In a preferred embodiment, the silk fibroin fiber matrix is ​​formed by self-assembly induced in a silk fibroin solution with a concentration ≥0.1 mg / mL at 0-10°C for ≥1 hour. Preferably, the concentration of the silk fibroin solution is ≥3 mg / mL, and more preferably, the concentration of the silk fibroin solution is ≥5 mg / mL. Preferably, self-assembly takes 1-3 days. The induction is carried out in a low-temperature environment, with an induction temperature of 0-10°C, preferably 4-8°C.

[0091] In a preferred embodiment, the crosslinking of the silk fibroin fiber matrix includes chemical crosslinking and physical crosslinking. In chemical crosslinking, the two aldehyde groups of glutaraldehyde form Schiff bases with the amino groups of the silk fibroin fiber matrix, connecting the silk fibroin fiber matrix with five-carbon bridges. In physical crosslinking, the silk fibroin fiber matrix is ​​subjected to severe dehydration (hydrothermal treatment) under high temperature and pressure to form physical crosslinks. During high temperature and pressure crosslinking, the crosslinking temperature is ≥80°C, the crosslinking pressure is ≥15 psi, and the crosslinking time is ≥15 min. Most preferably, the crosslinking temperature is 122.7°C and the crosslinking pressure is 22.6 psi, and the resulting crosslinked silk fibroin fiber mineralized matrix has a stable silk fibroin structure. The crosslinked silk fibroin fiber mineralized matrix will not dissolve or separate in the mineralization solution.

[0092] In a preferred embodiment, the mineralizing solution comprises a calcium salt, a stabilizer, and a phosphate. The calcium salt is a water-soluble calcium salt, selected from one or more of calcium chloride and calcium nitrate. The calcium ion concentration in the mineralizing solution is 0.2-20 mmol / L; more preferably 1-10 mmol / L. The stabilizer is selected from one or more of polyaspartic acid, polyaspartate, polyacrylic acid, and polyacrylate. When the stabilizer is polyaspartate, the polyaspartate is selected from one or more of potassium polyaspartate and sodium polyaspartate. When the stabilizer is polyaspartic acid, the concentration of polyaspartic acid is 10-400 μg / mL. Preferably, the concentration of polyaspartic acid is 50-200 μg / mL. More preferably, it is 50 μg / mL. When the stabilizer is polyaspartic acid, the molecular weight of polyaspartic acid is 3000-50000 g / mol. Preferably, the molecular weight of polyaspartic acid is 5000-50000 g / mol. More preferably, the molecular weight of polyaspartic acid is 20000-50000 g / mol. The phosphate is selected from one or more of disodium hydrogen phosphate and dipotassium hydrogen phosphate; the concentration of phosphate ions in the mineralization solution is 0.3-30 mmol / L, preferably 1-5 mmol / L.

[0093] In a preferred embodiment, the mineralization solution is prepared by mixing a calcium salt-polyaspartic acid solution with a phosphate solution. The mineralization solution is prepared by dissolving the calcium salt and polyaspartic acid in a Tris buffer solution at pH 7.4-7.8 (37°C), and then mixing it with a phosphate solution dissolved in the same Tris buffer solution. In this invention, the method described above allows the polyaspartic acid to first bind to calcium ions. After mixing with the phosphate ion solution, the calcium ions are slowly released, gradually forming amorphous calcium phosphate nanoclusters that preferentially penetrate into the silk fibroin fiber, effectively inhibiting the rapid conversion of calcium phosphate into hydroxyapatite and its deposition on the fiber surface. This invention involves immersing the cross-linked silk fibroin fiber mineralization matrix in the mineralization solution for mineralization, resulting in the biomimetic mineralized silk fibroin scaffold material.

[0094] In a preferred embodiment, the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralization matrix is ​​≥10; more preferably ≥100. In this invention, when the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralization matrix is ​​within the above range, the amount of mineralizing solution is sufficient, thereby ensuring that calcium phosphate inorganic matter can preferentially deposit into the silk fibroin fiber matrix. The mineralization time is ≥24 hours; more preferably ≥3 days. Preferably, the mineralizing solution is replaced every 1-3 days. After the mineralization is completed, the obtained product is washed with deionized water and freeze-dried to obtain the biomimetic mineralized silk fibroin scaffold material.

[0095] In a preferred embodiment, the silk fibroin is prepared by degumming, rinsing, drying, dissolving, dialysis purification, and centrifuging to remove impurities from silk cocoons to obtain silk fibroin.

[0096] Example 1

[0097] I. Preparation

[0098] (1) Preparation of silk fibroin

[0099] 5g of dried silkworm cocoons, cut into small pieces, were boiled in 2 liters of 0.02M Na₂CO₃ aqueous solution for 30 minutes to degummify, and then thoroughly rinsed with distilled water. The cocoons were dried in a fume hood for 24 hours. The dried fibroin was then dissolved in 9.3M lithium bromide solution at a weight-to-volume ratio of 1:4. After 4 hours at 60°C, the completely dissolved fibroin was dialyzed against 1L of deionized water for two days using a dialysis tube (cellulose, MWCO₃ kDa). The deionized water was changed every 8 hours. After dialysis, the fibroin solution was transferred to a 50ml centrifuge tube and centrifuged at 9000 RPM for 30 minutes at 4°C to remove impurities.

[0100] (2) Preparation of silk fibroin fibers

[0101] The silk fibroin solution obtained in step (1) was transferred to a new tube and placed in a refrigerator at 4°C for 24 hours to induce the silk fibroin to self-assemble into a fiber structure. The induced silk fibroin fiber matrix solution was poured into a Teflon mold, frozen with liquid nitrogen, and then freeze-dried for 24 hours to prepare a pure freeze-dried silk fibroin fiber matrix. Freeze-drying can effectively lock the morphology of the silk fibroin fiber matrix and prevent it from further self-assembling into a membrane structure.

[0102] (3) Preparation of cross-linked silk fibroin fiber mineralization matrix

[0103] The freeze-dried silk fibroin matrix from step (2) was sealed in an autoclave and then placed in a steam sterilizer. Physical cross-linking was performed at 122.7°C and 22.6 psi for 15 minutes.

[0104] (4) Preparation of biomimetic mineralized silk fibroin fiber scaffold materials

[0105] Tris buffer solution (pH 7.4, 37℃) was prepared by mixing 5.72g Trizma HCl, 1.66g Trizma Base, and 9g NaCl in 1L of deionized water. This buffer solution allows polyaspartic acid to bind calcium ions, and when mixed with phosphate ion solution, it allows calcium ions to be released slowly, forming amorphous calcium phosphate nanoclusters that preferentially penetrate into the silk fibroin fiber, effectively inhibiting the rapid conversion of calcium phosphate into hydroxyapatite and its deposition on the fiber surface. 9mM CaCl2 and 4.2mM K2HPO4 were added to the Tris buffer solution to prepare the calcium and phosphorus solutions required for biomimetic mineralization. Polyaspartic acid was dissolved in CaCl2 solution as a mineralization stabilizer to a concentration of 100μg / mL. Equal volumes of calcium ion solution and phosphate ion solution were mixed to prepare a mineralization solution. The cross-linked silk fibroin fiber matrix obtained in step (3) was placed in the mineralization solution and mineralized at 37℃, with the mineralization solution being replaced every 3 days. After mineralization, the material was thoroughly washed with deionized water and then freeze-dried to obtain a biomimetic mineralized silk fibroin scaffold material.

[0106] II. Results Analysis

[0107] (1) Secondary structure analysis of ATR-FTIR

[0108] This embodiment uses ATR-FTIR to detect structural changes in silk fibroin samples after different treatments. For example... Figure 1 As shown, fresh silk fibroin (Fresh) at 1646 cm⁻¹ -1 A peak appears at this point, exhibiting a characteristic random coil conformation. Silk fibroin induced to self-assemble at 4°C for 1 day reaches a peak value of 1621 cm⁻¹. -1 and 1700cm -1It reaches its peak at [a certain point] and exhibits characteristics of an antiparallel β-sheet structure. High-temperature, high-pressure crosslinking treatment further reduces the random coil conformation.

[0109] To analyze the content of secondary structure of silk fibroin, samples were taken from 1600-1700 cm⁻¹. -1 Quantitative analysis of the infrared spectrum within the region was performed, and the results are as follows: Figure 2 As shown in Table 1, fresh silk fibroin consists of random coil (RC) structures, β-sheets, and β-turns (T). After the silk fibroin solution was induced to self-assemble at 4°C for one day, the content of random coil (RC) structures decreased from 70.0% to 65.7%, while the content of β-sheet structures increased from 9.2% to 34.3%. The disappearance of β-turns (T) indicates that the increase in β-sheet structures is mainly due to β-turn transformation. High-temperature and high-pressure treatment of the silk fibroin matrix further increased the content of β-sheets.

[0110] Table 1. Peak positions and integral intensities of deconvolution amide I in silk fibroin

[0111]

[0112]

[0113] (2) Analysis of silk fibroin before and after self-assembly

[0114] The morphological characteristics of silk fibroin before and after self-assembly were observed using scanning electron microscopy. Fresh silk fibroin exhibits a bead-like morphology. Figure 3 As shown in A. (As indicated by...) Figure 3 As shown in Figure B, after induced self-assembly at 4°C for one day, silk fibroin formed interconnected fibers with a diameter of 200-500 nm. These silk fibroin fibers were uniformly distributed, forming a nonwoven fiber morphology. Through comparison... Figure 3 A and Figure 3 B, it can be concluded that the fibers in the silk fibroin fiber matrix described in this invention are formed from coagulated fibrin bead-like particles. For example... Figure 3 As shown in C, after high temperature and high pressure crosslinking, no obvious morphological changes were observed in the silk fibroin fiber matrix. The silk fibroin fiber matrix is ​​insoluble in water and can be used as a stable mineralization matrix.

[0115] (3) Morphological study of biomimetic mineralization in silk fibroin fibers

[0116] The cross-linked silk fibroin fiber mineralization matrix was mineralized in a mineralization solution for 3 days. For example... Figure 4 As shown in A, C, and D, the mineralized biomimetic mineralized silk fibroin scaffold material retains the morphology of nonwoven fibers. Compared to the unmineralized cross-linked silk fibroin fiber mineralized matrix, the fiber surface and inner side become rougher. Figure 4As shown in B, EDS elemental analysis identified high levels of calcium and phosphorus.

[0117] like Figure 5 As shown, ATR-FTIR spectral analysis of the mineralized matrix of cross-linked silk fibroin fibers before and after mineralization revealed that at 1031 cm⁻¹... -1 The presence of strong absorption bands is attributed to the vibrational modes of phosphate. These results indicate that hydroxyapatite minerals are embedded within the cross-linked silk fibroin fiber mineralized matrix, forming intra-matrix mineralization. This intra-matrix mineralization of cross-linked silk fibroin fibers replicates the composite structure of hydroxyapatite within collagen fibers in natural bone, achieving a high degree of biomimicry and promoting bone tissue regeneration and repair.

[0118] (4) Biomimetic morphology study of non-fibrous mineralized matrix of silk fibroin after mineralization

[0119] When silk fibroin self-assembles at low temperature for more than 3 days, the fibroin fiber morphology disappears, forming a membrane-like structure. For example... Figure 6 The image shown is a SEM image of a silk fibroin membrane matrix that has self-assembled at 4℃ for 5 days and then undergone biomimetic mineralization for 3 days. It can be seen that after 5 days of self-assembly, the fibrous morphology of the silk fibroin disappears, and calcium phosphate deposits only in localized areas of the mineralized silk fibroin matrix. This uneven mineralization deposition means that the mineralized silk fibroin material cannot reproduce the microstructure of natural bone.

[0120] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a biomimetic mineralized silk fibroin scaffold material, characterized in that, Includes the following steps: 1) Silk fibroin was induced to self-assemble in solution to obtain a silk fibroin fiber matrix; 2) Crosslink the silk fibroin fiber matrix obtained in step 1) to obtain a crosslinked silk fibroin fiber mineralized matrix; 3) The cross-linked silk fibroin fiber mineralized matrix obtained in step 2) is mineralized in a mineralizing solution to obtain the biomimetic mineralized silk fibroin scaffold material; In step 1), the temperature for inducing self-assembly is 4-8℃; In step 1), the induced self-assembly time is 1-3 days; In step 2), before the crosslinking, the silk fibroin fiber matrix obtained in step 1) is freeze-dried; In step 1), the concentration of the silk fibroin is ≥3 mg / mL; In step 3), the mineralization time is ≥24 hours; In step 3), the mineralization solution is prepared by mixing a first mixture comprising calcium salt, stabilizer and buffer solution and a second mixture comprising phosphate and buffer solution to obtain the mineralization solution; B1) The pH of the buffer solution is 7.4-7.8; B2) The buffer solution is a Tris buffer solution; B3) The buffer solution includes one or more of Trizma HCl, Trizma Base, and NaCl; In step 1), the solution is water.

2. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to claim 1, characterized in that, Includes at least one of the following technical features: 14) In step 1), the silk fibroin is derived from silkworm silk; 22) In step 2), the crosslinking is selected from at least one of chemical crosslinking and physical crosslinking; 32) In step 3), the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralizing matrix is ​​≥1; 34) In step 3), after mineralization, the process also includes washing and freeze-drying.

3. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to claim 2, characterized in that, Includes at least one of the following technical features: In feature 22), when the crosslinking is chemical crosslinking, the silk fibroin fiber matrix is ​​crosslinked with a crosslinking agent; 222) In feature 22), when the crosslinking is physical crosslinking, the silk fibroin fiber matrix is ​​dehydrated to obtain a crosslinked silk fibroin fiber mineralized matrix; 223) In feature 22), when the crosslinking is physical crosslinking, the temperature of the physical crosslinking is ≥80℃; 224) In feature 22), when the crosslinking is physical crosslinking, the pressure of the physical crosslinking is ≥15 psi; 225) In feature 22), when the crosslinking is physical crosslinking, the time of physical crosslinking is ≥15 min; 311) The calcium salt is a water-soluble calcium salt; 312) The calcium ion concentration in the mineralization solution is 0.2-20 mmol / L; 313) The stabilizer is selected from one or more of polyaspartic acid, polyaspartic acid salt, polyacrylic acid, and polyacrylate; 314) The phosphate is selected from one or more of disodium hydrogen phosphate and dipotassium hydrogen phosphate; The concentration of phosphate ions in the mineralization solution is 0.3-30 mmol / L; 321) In feature 32), the volume ratio of the mineralizing solution to the cross-linked silk fibroin fiber mineralizing matrix is ​​≥100; 331) In feature 3), the mineralization time is ≥3 days; 341) In feature 34), the reagent used for cleaning is deionized water.

4. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to claim 3, characterized in that, Includes at least one of the following technical features: In feature 221), the crosslinking agent is selected from one or more of glutaraldehyde, carbodiimide, and genipin; In feature 311), the calcium salt is selected from one or more of calcium chloride and calcium nitrate; In feature 312), the calcium ion concentration is 1-10 mmol / L; In feature 313), when the stabilizer is a polyaspartic acid salt, the polyaspartic acid salt is selected from one or more of potassium polyaspartic acid and sodium polyaspartic acid; In feature 313), when the stabilizer is polyaspartic acid, the concentration of polyaspartic acid is 50-200 µg / mL; In feature 313), when the stabilizer is polyaspartic acid, the molecular weight of polyaspartic acid is 3000-50000 g / mol; In feature 314), the concentration of the phosphate ions is 1-5 mmol / L.

5. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to any one of claims 1-4, characterized in that, In step 1), the silk fibroin is prepared by degumming, rinsing, drying, dissolving, dialysis purification, and centrifuging to remove impurities, thereby obtaining a silk fibroin solution.

6. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to claim 5, characterized in that, Includes at least one of the following technical features: A1) The degumming solution is a Na2CO3 solution; A2) The degumming method described is boiling degumming; A3) The degumming time is 30-60 minutes; A4) The rinsing solution is distilled water; A5) The drying time is 24-72 hours; A6) The solution to be dissolved is a lithium bromide solution; A7) The dialysis solution is deionized water; A8) The dialysis solution is used for 8-12 hours each time; A9) The dialysis time is 2-3 days; The centrifugation temperature described in A10 is 4-8℃; The centrifugation speed described in A11) is 6000-9000 RPM; The centrifugation time described in A12 is 5-30 min.

7. The method for preparing the biomimetic mineralized silk fibroin scaffold material according to claim 6, characterized in that, Includes at least one of the following technical features: In feature A1), the concentration of the Na2CO3 solution is 0.02-0.1M; In feature A2), the boiling degumming temperature is ≥90℃; In feature A6), the weight-volume ratio of the dried silk fibroin to the lithium bromide solution is 1:4 to 1:

8. In feature A6), the concentration of the lithium bromide solution is 9.3-9.5 M.

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