Preparation and modification method of high-strength and high-toughness silk protein artificial ligament

Abstract

This invention discloses a method for preparing and modifying high-strength, high-toughness silk fibroin artificial ligaments, belonging to the field of silk fibroin artificial ligaments. The method involves first blending regenerated silk fibroin with a polyketide solution, spinning the resulting fibers, and weaving them into an artificial ligament scaffold. The scaffold is then impregnated in a reactive impregnation solution prepared from a tannic acid-ferric chloride coordination mother liquor, a methyltrimethoxysilane pre-hydrolyzed mother liquor, and a Tris-HCl buffer solution. The reaction is carried out at pH 8.0-9.0 and 20-60°C for 12-24 hours. After washing, drying, and sterilization, the final product is obtained. This invention, through the synergistic effects of polyketide microcrystals, silk fibroin β-sheets, and tannic acid-ferric chloride, endows the material with high strength, high toughness, and excellent biostability. Degradation is controllable, the process is simple, and it is suitable for long-term implantation in vivo, providing an ideal material for soft tissue repair.

Description

Technical Field

[0001] This invention relates to the field of silk fibroin artificial ligaments, and particularly to a method for preparing and modifying a high-strength, high-toughness silk fibroin artificial ligament. Background Technology

[0002] With the increasing prevalence of sports injuries and soft tissue degeneration, ligament injuries have become a common clinical challenge. As an important material for repairing and replacing damaged ligaments, artificial ligaments must be designed with not only high strength and good toughness, but also excellent biocompatibility and controllable degradation to support the regeneration and functional recovery of damaged tissues.

[0003] Silk fibroin, a natural fibrous protein derived from silkworm silk, is composed of high-molecular-weight polypeptide chains. These chains are arranged with alternating β-sheet crystals and amorphous regions, providing both significant mechanical strength and flexibility. Simultaneously, silk fibroin contains abundant natural amino acid residues, endowing it with excellent biocompatibility, biodegradability, and low immunogenicity. It effectively supports cell adhesion, proliferation, and differentiation, providing an ideal basic material for constructing ligament replacement scaffolds. Silk fibroin can be processed into various forms, such as membranes, nanofibers / microfibers, gels, sponges, or three-dimensional porous scaffolds, through regeneration and recombination technologies to meet different tissue engineering needs. Its microstructure can be controlled through wet spinning, directional freeze-drying, and nanofiberization to enhance the material's strength and toughness. In load-bearing tissue engineering such as the anterior cruciate ligament (ACL), silk fibroin scaffolds exhibit unique advantages, serving as soft tissue substitutes and matrix materials for artificial ligaments. They can also be combined with other biopolymers or inorganic materials to improve tissue integration and functional performance.

[0004] In the existing technology, the preparation process of silk protein artificial ligaments is complex, involving multiple spinning, cross-linking and structural control operations. The process parameters have a significant impact on the final performance and it is difficult to completely match the mechanical properties of natural human ligaments. At the same time, there is still an incomplete coordination between its degradation rate and mechanical maintenance, which limits its long-term performance and reliability in clinical applications. Summary of the Invention

[0005] The main objective of this invention is to provide a method for preparing and modifying high-strength, high-toughness artificial ligaments made of silk protein, which can effectively solve the problems mentioned in the above claims.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing a high-strength, high-toughness artificial ligament made of silk protein includes the following steps: S1: Silk protein is mixed with polyketide solution to form a spinning solution, which is then used to prepare composite fibers through spinning process. The composite fibers are then woven into shapes to obtain an artificial ligament scaffold. S2: The artificial ligament scaffold prepared in step S1 is immersed in a reactive impregnation solution and reacted under pH conditions of 8.0-9.0 to obtain silk protein artificial ligament.

[0007] Preferably, the mass ratio of silk fibroin to polyketide added in step S1 is (7-9):(1-3); wherein the silk fibroin is regenerated silk fibroin.

[0008] Preferably, the treatment in step S2 involves removing the reacted artificial ligament and sequentially washing, drying, and sterilizing it; the sterilization treatment is ethylene oxide sterilization or cobalt-60 irradiation sterilization.

[0009] Preferably, the reaction temperature in step S2 is 20-60℃ and the reaction time is 12-24 hours.

[0010] Preferably, the specific preparation steps of the reactive impregnation solution in step S2 are as follows: A1: Dissolve tannic acid in deionized water, and add ferric chloride solution dropwise while stirring until the solution changes from light yellow to dark blue to obtain mother liquor A; A2: Pre-hydrolyze with methyltrimethoxysilane at pH 4-5 for 30-60 minutes to obtain mother liquor B; A3: Add the prepared stock solution A to the Tris-HCl buffer solution and stir until homogeneous. At the same time, add the stock solution B and stir vigorously at 500-800 rpm. Adjust the pH to 8.0-8.5 with NaOH solution to obtain the reactive impregnation solution.

[0011] Preferably, the mass ratio of tannic acid to ferric chloride added in step A1 is 1:(0.1-0.3).

[0012] Preferably, a crosslinking agent is added in step A2; the crosslinking agent is either glutaraldehyde or genipin.

[0013] Preferably, the volume ratio of mother liquor A to mother liquor B added in step A3 is 1:(0.2-0.5).

[0014] A method for modifying a high-strength and high-toughness silk protein artificial ligament includes the following steps: immersing a woven artificial ligament scaffold in a reactive impregnation solution and reacting it for 12-24 hours at pH=8.0-9.0 and a temperature of 20-60℃; then removing it and washing, drying, and sterilizing it to obtain the modified silk protein artificial ligament.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The technical solution of this invention is composed of rigid polyketide microcrystals, silk fibroin β-sheets, a tannic acid-ferric chloride coordination network, and a polysiloxane covalent network through multiple interactions. The carbonyl groups on the polyketide molecular chain form hydrogen bonds with the amino / hydroxyl groups of the silk fibroin, constructing a strong and tough interface capable of effectively transferring stress. The polyphenolic structure of the tannic acid forms hydrogen bonds with the silk fibroin on one hand, and dynamically reversible coordination bonds with ferric chloride on the other. This structure provides basic mechanical support through the polyketide microcrystals, enabling the artificial ligament to withstand large tensile loads; simultaneously, energy dissipation is achieved through the breaking and recombination of dynamic coordination bonds, endowing the ligament with excellent toughness.

[0016] 2. In the technical solution of this invention, the tannic acid-ferric chloride coordination network structure reduces the risk of direct breakage of the main chain of the material under external force, thereby improving the tensile strength and fatigue life of the artificial ligament. The polysiloxane network formed by the hydrolysis and condensation of methyltrimethoxysilane has methyl groups on its side chains arranged on the fiber surface to form a hydrophobic barrier, which can effectively block the invasion of water molecules and slow down the water absorption and swelling of the hydrophilic segments of silk fibroin, so that the artificial ligament has excellent dimensional stability and mechanical retention in the physiological environment. The polysiloxane covalent network can also act as a physical protective layer, masking or buffering the direct attack of external enzymatic hydrolysis on the fiber matrix, making the artificial ligament more suitable for long-term implantation in the body. By adjusting the density of the tannic acid-ferric chloride coordination bonds or the crosslinking density of the polysiloxane, the release rate of bioactive molecules in the inflammatory microenvironment can be controlled, which helps to achieve the sustained anti-inflammatory effect of the implant and improve the long-term efficacy of tissue repair. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the preparation process of the present invention; Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise range thresholds, and these range thresholds should be understood to include values ​​close to these range thresholds. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0019] The following describes in detail, with reference to the accompanying drawings, a method for preparing and modifying a high-strength, high-toughness silk protein artificial ligament provided in the embodiments of this specification.

[0020] like Figure 1 The diagram shows a process flow chart for the preparation and modification of a high-strength, high-toughness artificial ligament made of silk protein.

[0021] Example 1 A method for preparing a high-strength, high-toughness artificial ligament made of silk protein, the specific implementation steps of which are as follows: S1: Dissolve 40g of regenerated silk fibroin in 460mL of deionized water to prepare an 8% (w / w) protein solution; dissolve 10g of polyetheretherketone in 73.3g of N-methylpyrrolidone (total mass 83.3g) to prepare a 12% (w / w) polymer solution; mix the two solutions in proportion and stir at 600rpm for 2 hours to form a uniform spinning solution; use a wet spinning process at a spinning temperature of 25℃ and a spinning speed of 15m / min; after coagulation in a coagulation bath (60g ethanol: 140g water, volume ratio 3:7, ethanol density 0.79g / mL, water 1g / mL), and wash with water to obtain composite fibers; braid the composite fibers using a three-strand braiding process (braiding density 12 strands / cm) to obtain an artificial ligament scaffold.

[0022] S2: Weigh 10g of tannic acid and 2g of ferric chloride (mass ratio 1:0.2). Dissolve 10g of tannic acid in 500mL of deionized water. While stirring, add 20g of 10% ferric chloride solution (containing 2g of ferric chloride) dropwise until the solution turns dark blue to obtain mother liquor A. Take 15 mL of methyltrimethoxysilane (approximately 14.7 g, density 0.98 g / mL), add 0.5 g of glutaraldehyde as a crosslinking agent, adjust the pH to 4.5 with hydrochloric acid, and pre-hydrolyze for 45 minutes to obtain mother liquor B; Take 50 mL of stock solution A and add it to 450 mL of Tris-HCl buffer (pH=7.4). After stirring evenly, add 15 mL of stock solution B (about 15.2 g) and stir vigorously at 700 rpm for 30 minutes. Adjust the pH to 8.2 with 1 mol / L NaOH solution (about 40 g / L) to obtain the reactive impregnation solution.

[0023] The artificial ligament scaffold was completely immersed in a reactive impregnation solution, with the reaction temperature controlled at 40℃ and pH=8.5 for 12 hours. After removal, it was washed with deionized water (100g each time, 3 times in total) until neutral, and then vacuum dried at 60℃ for 12 hours. The dried product weighed about 11.2g and was sterilized with ethylene oxide to obtain a high-strength and high-toughness silk protein artificial ligament.

[0024] Example 2 S1: Dissolve 35g of regenerated silk fibroin in 315mL of deionized water to prepare a 10% aqueous solution; dissolve 15g of polyketone in 85g of dimethylformamide (total mass 100g) to prepare a 15% polymer solution; mix the two solutions and stir at 700rpm for 2.5 hours; use electrospinning process with voltage 20kV, receiving distance 15cm, and spinning speed 20m / min to obtain composite fibers; braid using a four-strand braiding process (braiding density 15 strands / cm) to obtain an artificial ligament scaffold.

[0025] S2: Weigh 12g of tannic acid and 1.2g of ferric chloride (mass ratio 1:0.1), dissolve 12g of tannic acid in 600mL of deionized water, and add 12g of 10% ferric chloride solution (containing 1.2g of ferric chloride) to obtain mother liquor A; Take 20 mL of methyltrimethoxysilane (approximately 19.6 g, density 0.98 g / mL), add 0.8 g of genipin, adjust the pH to 4.0 with hydrochloric acid, pre-hydrolyze for 60 minutes to obtain mother liquor B (total mass approximately 20.4 g). Take 60 mL of stock solution A (approximately 62.4 g) and add it to 540 mL of Tris-HCl buffer (540 g). Add 12 mL of stock solution B (approximately 12.24 g), stir at 800 rpm for 40 minutes, and adjust the pH to 8.0 to obtain the reactive impregnation solution (total mass approximately 614.64 g).

[0026] The reaction was carried out at 60℃ and pH=9.0 for 24 hours. After washing (100g of deionized water each time, for a total of 3 times), the product was dried with hot air at 50℃ for 15 hours. The dried product weighed about 11.5g. The product was sterilized by cobalt-60 irradiation to obtain a high-strength and high-toughness silk protein artificial ligament.

[0027] Example 3 45g of regenerated silk fibroin was dissolved in 585mL of deionized water to prepare a 7% aqueous solution; 5g of polyetherketone was dissolved in 45g of N-methylpyrrolidone (total mass 50g) to prepare a 10% polymer solution; the two solutions were mixed and stirred at 500rpm for 1.5 hours; a melt spinning process was used with a spinning temperature of 280℃ and a spinning speed of 12m / min to obtain composite fibers; the fibers were then braided using a two-strand braiding process (braiding density 10 strands / cm) to obtain an artificial ligament scaffold.

[0028] S2: Weigh 8g of tannic acid and 2.4g of ferric chloride (mass ratio 1:0.3), dissolve 8g of tannic acid in 400mL of deionized water, and add 24g of 10% ferric chloride solution (containing 2.4g of ferric chloride) to obtain mother liquor A; Take 10 mL of methyltrimethoxysilane (approximately 9.8 g, density 0.98 g / mL), without any additional cross-linking agent, adjust the pH to 5.0 with hydrochloric acid, and pre-hydrolyze for 30 minutes to obtain mother liquor B (total mass approximately 9.8 g). Take 40 mL of stock solution A and add it to 360 mL of Tris-HCl buffer (360 g). Add 20 mL of stock solution B, stir at 500 rpm for 20 minutes, and adjust the pH to 8.5 to obtain the reactive impregnation solution.

[0029] The reaction was carried out at 20℃ and pH=8.0 for 18 hours. After washing (100g of deionized water each time, for a total of 3 times), the product was vacuum dried at 70℃ for 10 hours. The dried product weighed about 11.0g. The product was sterilized with ethylene oxide to obtain a high-strength and high-toughness silk protein artificial ligament.

[0030] Comparative Example 1 The difference from Example 1 is that in the reactive impregnation solution A1 step, 10g of tannic acid and 0.05g of ferric chloride are weighed out, while the rest of the steps are the same as in Example 1.

[0031] Comparative Example 2 The difference from Example 1 is that in the reactive impregnation solution A3 step, 50 mL of mother solution A is added to 450 mL of Tris-HCl buffer, and 5 mL of mother solution B is added. The volume ratio of mother solution A to mother solution B is 1:0.01. The remaining steps are the same as in Example 1.

[0032] Comparative Example 3 The difference from Example 1 is that the impregnation reaction temperature is adjusted to 70°C, pH=8.5, and the reaction time is 12 hours, while the remaining steps are the same as in Example 1.

[0033] Comparative Example 4 The difference from Example 1 is that in the reactive impregnation solution A2 step, the pre-hydrolysis time of methyltrimethoxysilane is adjusted to 20 minutes, while the other steps are the same as in Example 1.

[0034] The artificial ligaments prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to the following performance tests: 1. Adjust each group of samples to a length of 50mm, gauge length of 20mm, and width of 3mm. In an environment of 25℃ and relative humidity of 50%, use a universal testing machine to conduct tensile tests at a tensile speed of 10mm / min. Test 5 samples in each group. Clamp both ends of the sample in the upper and lower clamps of the testing machine to ensure that the sample is vertical, without twisting or loosening. The clamping position is 10mm away from both ends of the sample. Take the average value of the test results and record the three indicators of breaking strength, elongation at break, and elastic modulus.

[0035] 2. In vitro degradation experiments used PBS buffer (pH 7.4). The dried samples were weighed and recorded as M0. Each sample was placed in a 50 mL centrifuge tube, and 40 mL of simulated body fluid (SBF, pH 7.4) was added to ensure complete immersion. The centrifuge tube caps were tightened, and the tubes were placed in a constant-temperature water bath at 37°C (simulating human body temperature) for static soaking. During soaking, the simulated body fluid was changed every 3 days. Each time the simulated body fluid was changed, the sample surface was gently rinsed with deionized water, the surface moisture was blotted dry with filter paper, and the mass was quickly measured and recorded. The sample was immediately returned to the new simulated body fluid for further soaking. When changing the simulated body fluid, the pH of the newly prepared SBF was checked with a pH meter to ensure it remained at 7.4. After 30 days of soaking, all samples were removed, rinsed three times repeatedly with deionized water to remove any residual simulated body fluid, and placed in a vacuum drying oven at 60°C for 12 hours. After cooling to room temperature, the remaining mass of the sample was accurately weighed (recorded as m1). The experimental data are shown in Table 1-2.

[0036] The in vitro degradation rate of each group of samples was calculated using the formula: Degradation rate (%) = (m0 - m1) / m0 × 100% Table 1: Mechanical property test results of Examples 1-3 and Comparative Examples 1-4 Table 2: In vitro degradation rate test results of Examples 1-3 and Comparative Examples 1-4 As shown in Table 1, the artificial ligaments prepared in Examples 1-3 all exhibited excellent performance in terms of fracture strength, elongation at break, and elastic modulus. Among them, Example 1, using a wet spinning process, showed the best performance, indicating that the silk fibroin / polyketone composite fiber scaffold, after being modified with an optimized ratio of tannic acid-ferric chloride coordination network and polysiloxane covalent network, can form an ideal multi-synergistic structure of "high-strength skeleton + dynamic toughening + hydrophobic protection," effectively simulating the "high strength and high toughness" mechanical properties of natural ligaments. In Comparative Example 1, the TA-Fe³⁺ ratio was severely imbalanced (1:0.005), resulting in insufficient density of the dynamic coordination network and failure of the sacrificial bond toughening mechanism, thus leading to the worst mechanical properties. This demonstrates that the coordination network is the key source of toughness. In Comparative Example 2, the amount of polysiloxane precursor (mother liquor B) added was too low, resulting in a severe deficiency of the hydrophobic crosslinking network, weakening the internal support and protection of the material and also resulting in poor mechanical properties. Comparative Examples 3 and 4 may have had their fiber structure damaged or their crosslinking network integrity affected due to excessively high reaction temperature and excessively short silane pre-hydrolysis time, respectively. Their mechanical properties were between those of the Examples and Comparative Examples 1-2, further demonstrating that each process parameter has a significant impact on the final performance.

[0037] As shown in Table 2, the degradation rates of Examples 1-3 after immersion in simulated body fluids for 30 days (8.84%-11.06%) were significantly lower than those of all comparative examples, indicating their excellent in vitro stability. This is mainly attributed to the hydrophobic protective layer of polysiloxane formed by the hydrolysis and condensation of methyltrimethoxysilane, which effectively blocks water penetration and enzyme attack, delaying the degradation of the silk fibroin matrix. Example 1 had the lowest degradation rate, possibly related to its most ideal polysiloxane network formation. Comparative Examples 1 and 2 had the highest degradation rates (22.75% and 20.55%, respectively), due to insufficient dynamic coordination network and the absence of a hydrophobic protective layer, resulting in a loose material structure and poor erosion resistance. The degradation rates of Comparative Examples 3 and 4 (18.18% and 12.96%, respectively) were higher than those of Examples 1-3, indicating that improper reaction temperature and insufficient pre-hydrolysis can also impair the stability of the composite material, reducing its ability to maintain its structure in physiological environments.

[0038] In summary, this invention successfully constructs a composite material with multi-network synergistic reinforcement by spinning regenerated silk fibroin and polyketide into an artificial ligament scaffold, and then modifying it with a "reactive impregnation solution" composed of a TA-Fe³⁺ coordination network and a polysiloxane covalent network. Data from Examples 1-3 show that the artificial ligaments prepared by this method achieve good results in simulating the high strength, high toughness, mechanical properties, and long-term implantation stability of human ligaments. The performance degradation in the comparative examples, from the opposite perspective, verifies the effectiveness of TA-Fe... 3+ The coordination network, polysiloxane network, and key process parameters are all indispensable for achieving the "high strength, high toughness, and stability" goals of this invention. This method provides an effective approach for developing high-performance implantable soft tissue repair materials.

[0039] In the description of this specification, the reference to terms such as "embodiment," "various embodiments," etc., indicates that a specific feature, structure, material, or characteristic described in connection with that embodiment or preparation example is included in at least one embodiment of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments.

[0040] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a high-strength, high-toughness artificial ligament made of silk protein, characterized in that, Includes the following steps: S1: Silk protein is mixed with polyketide solution to form a spinning solution, which is then used to prepare composite fibers through spinning process. The composite fibers are then woven into shapes to obtain an artificial ligament scaffold. S2: The artificial ligament scaffold prepared in step S1 is immersed in a reactive impregnation solution and reacted under pH conditions of 8.0-9.0 to obtain silk protein artificial ligament.

2. The method for preparing high-strength, high-toughness silk fibroin artificial ligament according to claim 1, characterized in that, The mass ratio of silk protein to polyketide added in step S1 is (7-9):(1-3). The silk protein in this product is regenerated silk fibroin.

3. The method for preparing high-strength, high-toughness silk fibroin artificial ligament according to claim 1, characterized in that, The process in step S2 involves removing the reacted artificial ligament and sequentially washing, drying, and sterilizing it; the sterilization process is either ethylene oxide sterilization or cobalt-60 irradiation sterilization.

4. The method for preparing high-strength, high-toughness silk fibroin artificial ligament according to claim 1, characterized in that, In step S2, the reaction temperature is 20-60℃ and the reaction time is 12-24 hours.

5. The method for preparing a high-strength, high-toughness artificial ligament according to claim 1, characterized in that, The specific preparation steps of the reactive impregnation solution in step S2 are as follows: A1: Dissolve tannic acid in deionized water, and add ferric chloride solution dropwise while stirring until the solution changes from light yellow to dark blue to obtain mother liquor A; A2: Pre-hydrolyze with methyltrimethoxysilane at pH 4-5 for 30-60 minutes to obtain mother liquor B; A3: Add the prepared stock solution A to the Tris-HCl buffer solution and stir until homogeneous. At the same time, add the stock solution B and stir vigorously at 500-800 rpm. Adjust the pH to 8.0-8.5 with NaOH solution to obtain the reactive impregnation solution.

6. The method for preparing a high-strength, high-toughness artificial ligament according to claim 5, characterized in that, In step A1, the mass ratio of tannic acid to ferric chloride is 1:(0.1-0.3).

7. The method for preparing a high-strength, high-toughness artificial ligament according to claim 5, characterized in that, A cross-linking agent is added in step A2; the cross-linking agent is either glutaraldehyde or genipin.

8. The method for preparing a high-strength, high-toughness artificial ligament according to claim 5, characterized in that, In step A3, the volume ratio of mother liquor A to mother liquor B is 1:(0.2-0.5).

9. A method for modifying a high-strength, high-toughness silk protein artificial ligament, characterized in that, Includes the following steps: The woven artificial ligament scaffold is immersed in a reactive impregnation solution and reacted for 12-24 hours at pH=8.0-9.0 and temperature of 20-60℃. After removal, it is washed, dried and sterilized to obtain the modified silk protein artificial ligament.

Images