Preparation method of high-toughness silk fibroin hydrogel

Abstract

The application provides a preparation method of a high-strength and high-biocompatibility silk fibroin hydrogel, which utilizes a natural silk fibroin material, and realizes the improvement of the maximum tensile strength of the silk fibroin hydrogel support and the formation of a micro directional pore structure through a directional freezing and heating coaxial stretching processing method, wherein the heating temperature needs to be higher than the glass transition temperature of the silk fibroin material, so that a mechanical reinforced hydrogel support with good biocompatibility is finally obtained. The rotator cuff patch prepared by using the hydrogel support can achieve a similar mechanical strength to the rotator cuff tendon in the human body, has good biocompatibility, and has a micro directional pore with a micron scale in the micro morphology, which is beneficial to the directional growth of cells on the material surface and inside, and can realize a higher repair efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogel preparation, in particular to a preparation method of high-toughness silk fibroin hydrogel, and especially to a preparation method of high-toughness silk fibroin hydrogel which can be used as a rotator cuff patch, an anterior cruciate ligament substitute and the like. BACKGROUND

[0002] Tendon injury is one of the common sports injuries. Natural tendon is mainly composed of dense type I collagen, has a low cell content and weak angiogenic ability, and thus is difficult to heal after injury. Patients with large-scale tendon injury such as large rotator cuff tear and anterior cruciate ligament tear often need to be treated with a patch. At present, there are many tendon patch preparation methods. Traditional technologies such as autologous material and allogeneic material patches have problems such as insufficient source and easy to cause complications in the donor area. Existing artificial patches have the disadvantages of low mechanical strength of synthetic material scaffolds and strong immunogenicity. These shortcomings result in poor tendon repair effect, and thus it has become a research hotspot to find a patch material with excellent mechanical properties and biocompatibility.

[0003] Silk fibroin is widely used in the preparation of tissue engineering scaffolds, has good biocompatibility, degradability and superior mechanical properties. Silk fibroin is abundant in source and can be extracted from silk. Silk has a unique protein polymer structure, which has silk fibroin as the core and adheres sericin on the surface. By degumming the silk, silk fibroin can be obtained. Silk fibroin contains 18 kinds of amino acids, most of which are glycine, alanine and serine. Silk fibroin can be completely degraded in the body, and in the degradation process, the cell microenvironment is enriched with a large amount of amino acids, especially proline and lysine, which are the main raw materials for collagen synthesis. The repeated GAGAG sequence in silk fibroin can form a secondary structure of beta sheet through self-assembly, and this structure endows silk fibroin with excellent mechanical strength and toughness. It has been reported that the toughness of silk fibroin can even be superior to the best synthetic materials. At the same time, silk fibroin has adjustable mechanical properties. By adjusting the proportion of beta sheet structure in silk fibroin, the mechanical strength of silk fibroin material can be further improved and is superior to most currently commonly used degradable polymeric materials such as collagen and polylactic acid. In addition, the surface of silk material is easy to process and modify, and can be processed into various forms from liquid solution to organic solid state, such as gel, pipeline, hydrogel, microsphere, film and the like. These characteristics of silk material make it have broad prospects in tissue engineering applications. However, the mechanical strength of the existing silk fibroin is still difficult to reach the similar mechanical strength of the rotator cuff tendon in the human body, and thus it is urgent to find a silk fibroin with better mechanical properties. SUMMARY

[0004] To solve the above problems, the application provides a preparation method of high-toughness silk fibroin hydrogel which can be used as a tendon patch, utilizes natural silk fibroin material, realizes the improvement of the maximum tensile strength of the silk fibroin hydrogel scaffold through a processing method of directional freezing and heating coaxial stretching (higher than the glass transition temperature of the silk fibroin material), and has a micro directional pore structure, finally obtains a mechanically enhanced water material with good biocompatibility, can be used for preparing a tendon patch, can achieve similar mechanical strength to the rotator cuff tendon in the human body, has good biocompatibility and degradability, and the micro morphology has a micron-scale directional pore, is beneficial to the directional growth of cells on the material surface and inside, and can realize higher repair efficiency.

[0005] In one aspect, the application provides a preparation method of a hydrogel, mainly by directional freezing, freeze-drying and then stretching a silk fibroin solution.

[0006] The silk fibroin solution is prepared from natural silk degumming.

[0007] The directional freezing technology is to place the polymer solution in a temperature field to reduce the temperature, and as the temperature decreases, the solvent (usually water) gradually solidifies along the temperature gradient, and the ice crystal column produced by solidification extrudes, directional rearranges and embeds the polymer chain between the ice crystal columns. During this process, the ice crystal acts as an orientation template for the polymer chain, and plays a role of physical confinement. Directional freezing can form an anisotropic structure, so that the material has different orientations in space, and the mechanical properties in the oriented direction are much stronger than before directional freezing; in addition, due to the occupation of ice crystals, the local silk fibroin molecular aggregation degree will be increased, further enhancing the mechanical properties. Compared with ordinary silk fibroin hydrogel without directional freezing, the maximum tensile stress of the directional freezing silk fibroin hydrogel can be enhanced by several times. In addition, through the directional freezing technology, the silk fibroin solution can form a parallel pipe structure with 5-10um parallel pores, which is similar to the microstructure of the tendon. This microstructure can guide the stimulation of tendon stem cells to differentiate into tendon cells on the one hand, and is beneficial to the infiltration and directional arrangement of cells inside the scaffold on the other hand, thereby promoting the efficiency and effect of tendon regeneration.

[0008] Then, the silk fibroin material is subjected to heating coaxial stretching, and the heating temperature is higher than the glass transition temperature of the silk fibroin material, and the mechanical properties of the material can be greatly improved through heating coaxial stretching.

[0009] Coaxial stretching is to give a certain strain to the material, and the material is stretched to a certain length. The molecules in the material will rearrange under the action of the generated tensile stress, so that the material has a certain orientation structure in the stretching direction, and the tensile strength in the orientation is enhanced. In addition, amorphous polymers have three mechanical states, which are glassy, high-elastic and viscous flow state. At a lower temperature, the material is rigid and solid, similar to glass, and only a very small deformation will occur under the action of external force, which is the glassy state. When the temperature continues to rise to a certain range, the deformation of the material increases significantly, and the deformation is relatively stable in a certain temperature interval, which is the high-elastic state. The glass transition temperature (Tg) refers to the temperature corresponding to the transition from the glassy state to the high-elastic state. Silk is in the glassy state at room temperature, and only a small deformation can occur during coaxial stretching. Heating above Tg makes silk enter the high-elastic state, the movement of silk molecules is intensified, and the activity range is increased. The silk material in this state is more prone to deformation, and can be stretched to a higher ratio during coaxial stretching. The intensified molecular movement is more conducive to the formation of molecular orientation.

[0010] The glass transition temperature of the dried silk fibroin film is about 178℃, and in the presence of water molecules as a plasticizer, the glass transition temperature can be reduced to 77℃ under the conditions adopted by the application, greatly simplifying the operation difficulty and reducing the cost.

[0011] The coaxial stretching of the hydrated silk fibroin at different temperatures above Tg and different tensile strains can increase the maximum tensile stress of the silk fibroin hydrogel by about 10 times.

[0012] The directional freezing and heating stretching adopted by the application also have a significant synergistic effect, which increases the maximum tensile strength of the silk fibroin hydrogel by nearly 100 times, and also has better biocompatibility. The directional pore structure under the microscope is conducive to cell growth, and is more conducive to tendon injury repair.

[0013] The heating stretching must be performed after the directional freezing, and must be coaxial stretching along the direction of the directional freezing: 1. The directional freezing step is a prerequisite for the heating stretching. Direct stretching without directional freezing will cause the silk fibroin hydrogel to break directly; 2. If coaxial stretching is not performed along the direction of the directional freezing after the directional freezing, the silk fibroin hydrogel will also break directly.

[0014] Further, the preparation method comprises the following steps:

[0015] (1) Pouring the silk fibroin solution into a mold, directional freezing to obtain a silk ice block;

[0016] (2) Freeze-drying and cross-linking the silk ice block, and then soaking in water to hydrate to obtain a hydrogel;

[0017] (3) heating and stretching the hydrogel.

[0018] Further, the directional freezing in step (1) means that the freezing source is close to the mold, and the silk fibroin solution in the mold starts to generate ice crystals from the contact surface close to the freezing source and generates ice crystals in the direction away from the contact surface of the freezing source. The temperature of the freezing source is lower than -20℃.

[0019] Any device or product with a temperature-lowering effect can be used as the freezing source of the present application.

[0020] Further, step (1) is to pour the silk fibroin solution into the columnar space in the mold; place the mold above the metal rod, and immerse one side of the metal rod in liquid nitrogen.

[0021] In some ways, the metal rod as the freezing source is located below the mold and is in full contact with the bottom surface of the mold. When freezing, the ice crystals of the silk fibroin material grow upward from the bottom freezing source, thereby having a certain directionality, which is perpendicular to the bottom surface in contact with the freezing source, and then grows away from the freezing source.

[0022] In some ways, the metal rod as the freezing source is located on the side of the mold and is in full contact with the side of the mold. When freezing, the ice crystals of the silk fibroin material grow directionally from the side in contact with the freezing source, the direction of the ice crystal growth is perpendicular to the bottom surface of the freezing source, and then grows away from the freezing source.

[0023] In some ways, the metal rod as the freezing source is located on the top surface of the mold and is in full contact with the top surface of the mold. When freezing, the ice crystals of the silk fibroin material grow downward from the top surface in contact with the freezing source, the direction of the ice crystal growth is perpendicular to the bottom surface of the freezing source, and then grows away from the freezing source.

[0024] In some ways, step (1) is to pour the silk fibroin solution into the mold, and the mold has a columnar space of 4*4*20mm. The mold is placed above an aluminum rod, and the width of the aluminum rod can completely cover the contact surface with the mold. One side of the aluminum rod is immersed in liquid nitrogen. After 10 minutes, the mold is taken out, the internal silk fibroin ice block is taken out, and placed in a-80℃ refrigerator overnight.

[0025] Further, the cross-linking in step (2) is chemical cross-linking or enzyme cross-linking.

[0026] Since silk fibroin can be dissolved in water, it needs to be cross-linked to form a hydrogel. Cross-linking can make the silk fibroin scaffold stable in an aqueous solution and further improve the strength of the material.

[0027] It should be noted that the cross-linking process must be performed after the directional freezing is completed, because if the cross-linking is performed first, the molecular structure is fixed in advance, and cannot be oriented and arranged along with the growth of ice crystals during the directional freezing, so that the scaffold finally loses the directional structure.

[0028] The present application can adopt any method for cross-linking, such as ethanol cross-linking, other chemical cross-linking, enzyme cross-linking, etc., as long as the cross-linking of the silk fibroin material can be achieved, which is within the protection scope of the present application.

[0029] Further, the step (2) is to freeze-dry the silk fibroin ice block in a freeze-drying machine, and then soak it in anhydrous ethanol for cross-linking, and then soak it in water for sufficient hydration.

[0030] In some modes, the present application adopts anhydrous ethanol for cross-linking, and the silk fibroin ice block needs to be freeze-dried before cross-linking, because if the cross-linking is directly performed without freeze-drying, the internal microstructure of the silk fibroin ice block at low temperature will be destroyed before the cross-linking in ethanol, along with the melting of ice crystals. After the silk fibroin ice block is freeze-dried, a complete silk fibroin scaffold with a micro directional structure is obtained, and then the scaffold is placed in anhydrous ethanol for intermolecular cross-linking, to form a more stable silk fibroin hydrogel structure.

[0031] After cross-linking, the scaffold needs to be soaked in water for hydration, because the glass transition temperature of the dried silk fibroin material is about 178℃, and with the change of humidity, the glass transition temperature can be reduced to below 77℃ with water molecules as plasticizers. After the glass transition temperature is reduced, the operation difficulty of the heating and stretching is greatly simplified, and the cost is reduced. In addition, if the coaxial stretching is performed at a temperature higher than 100 degrees, the water in the hydrogel will evaporate quickly, resulting in the loss of water molecules as plasticizers, so that the hydrogel becomes brittle and the stretching length is limited.

[0032] Sufficient hydration means that the mass of the scaffold does not change after soaking in water for a long time, which indicates that the hydration is sufficient. Generally, the scaffold is soaked in water for more than 48 hours.

[0033] In some modes, the silk fibroin ice block can be freeze-dried in a freeze-drying machine for 72 hours to obtain a dried scaffold, and then soaked in anhydrous ethanol for cross-linking for 6 hours, and then soaked in water for 48 hours to ensure sufficient hydration.

[0034] Further, the heating and stretching in step (3) means heating to a temperature above the glass transition temperature of silk fibroin, and then performing coaxial stretching, and the direction of the coaxial stretching is consistent with the direction of the directional freezing.

[0035] The direction of the coaxial stretching is consistent with the direction of the directional freezing, so that the mechanical properties of the prepared scaffold are better, and there are directional micro-pores, which are beneficial to the growth of cells.

[0036] The hydrogel after directional freezing and drying and cross-linking has vertical stripes in appearance, and the direction of the micro-pore channels formed by directional freezing can be distinguished by naked eyes. At this time, heating to above the glass transition temperature of the silk fibroin is performed, and then directional coaxial stretching is performed in the same direction to rapidly improve the mechanical strength.

[0037] Further, step (3) is that the hydrogel after hydration is preheated at a temperature of 25-130℃, and then the material is stretched by 30-90% of the initial length.

[0038] In some modes, the temperature of heating only needs to be above the glass transition temperature of the silk fibroin, and the glass transition temperature of the silk fibroin hydrogel after hydration can be reduced to below 77℃, such as 25℃.

[0039] Further, step (3) is that the hydrogel after hydration is preheated at a temperature of 25-130℃ for 5 min, and then the material is stretched by 30-90% of the initial length and kept for 15 min, and then taken out and soaked in water to obtain the final hydrogel after hydration.

[0040] In some modes, the heating and stretching can be completed in a heating and stretching device, such as a high-temperature mechanical testing machine, as long as it can simultaneously heat and stretch.

[0041] In some modes, the pre-tension for heating and stretching only needs to be able to pull the hydrogel, so that the hydrogel does not have obvious wrinkles under the pre-tension.

[0042] In some modes, the ratio of heating and stretching is not fixed, as long as the material is stretched, the mechanical properties of the hydrogel can be improved, so the stretching can be performed according to the actual needs, and the scaffold can be ensured not to be pulled off. When the material is stretched by more than 90% of the initial length, part of the material may be broken.

[0043] In some modes, step (3) is that a pre-tension of 2N is given to the hydrogel after hydration, preheated at 95℃ for 5 min, and then the material is stretched by 30-90% of the initial length at a rate of 10 mm / min at 95℃ and kept for 15 min, and then taken out and soaked in water to obtain the final hydrogel after hydration.

[0044] Further, the preparation method of the silk fibroin solution in step (1) is as follows: after the silkworm pupae are removed from the silkworm cocoons, the silk is degummed by boiling in 0.02M sodium carbonate for 30 min, and the degummed silk fibers are dried in a fume hood for 12 h; dissolved in 9.3M lithium bromide at 60℃ for 4 h; the solution is dialyzed in a dialysis bag for 72 hours to remove lithium bromide; and the obtained solution is naturally air-dried in a fume hood for 6 h to obtain a silk fibroin solution with a concentration of 15%.

[0045] The present application utilizes natural silk, and prepares a silk fibroin solution after degumming treatment of the silk, performs directional freezing and freeze-drying on the silk fibroin solution, and then performs cross-linking by using anhydrous ethanol, so that the obtained scaffold is fully hydrated, and then stretched at a rate of 10 mm / min by 60% of the initial length under high-temperature conditions (95 DEG C) higher than the glass transition temperature, and kept for 15 minutes, and then the scaffold is taken out and further hydrated, so that the final hydrogel is obtained.

[0046] In another aspect, the present application provides a use of a method in improving the mechanical properties of a material, the method comprising directional freezing and freeze-drying of the material, and then coaxial heating and stretching of the material at a temperature higher than the glass transition temperature of the material; the material is PVA, gelatin, etc.

[0047] The present application provides a synergistic processing method of heating and stretching and directional freezing. The directional freezing can not only impart a directional pore structure to the material at a micro level, but also improve the mechanical properties of the material through the formation of the anisotropic structure. Further heating and stretching of the material at a temperature above the glass transition temperature can further greatly improve the mechanical properties of the material and the maximum tensile stress of the material. At the same time, the directional pore structure of the hydrogel at a micro scale can guide the differentiation of stimulated cells through the micro topography, and is also conducive to the penetration of cells on the scaffold and the acceleration of tissue repair.

[0048] The synergistic processing method of directional freezing and heating and stretching provided by the present application is not only suitable for silk fibroin materials, but also suitable for the improvement of the mechanical properties of other materials.

[0049] In another aspect, the present application provides a use of a silk fibroin hydrogel for preparing a rotator cuff patch or an anterior cruciate ligament substitute, the silk fibroin hydrogel being prepared by directional freezing, freeze-drying and then heating and stretching of a silk fibroin solution, and the heating temperature being higher than the glass transition temperature of the silk fibroin.

[0050] In summary, the present application provides a pre-protective processing method of stretching above the glass transition temperature (referred to as heating and stretching), and a synergistic processing method of heating and stretching and directional freezing, which are used for improving the mechanical properties of materials and obtaining the surface micro topography, and are conducive to the efficiency of tendon repair.

[0051] The silk fibroin hydrogel scaffold provided by the present application has the following beneficial effects:

[0052] 1. Excellent mechanical properties, the maximum stress of traditional silk hydrogel is about 0.02-0.2 MPa, and the present application can reach more than 10 MPa, which is improved by hundreds of times;

[0053] 2. The topological structure of the hydrogel surface can promote the directional arrangement of tendon stem cells;

[0054] 3. Good biocompatibility, the silk material itself has good biocompatibility, and no difficult-to-remove organic reagent is used in the preparation process of the hydrogel scaffold, so that the finally prepared scaffold has good biocompatibility;

[0055] 4. The silk material is widely sourced compared with autologous tendon, and the preparation process is simple;

[0056] 5. The provided directional freezing and heating stretching synergistic processing method is not only suitable for silk fibroin material, but also suitable for improving the mechanical properties of other materials. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 The preparation flow chart of the silk fibroin hydrogel scaffold is shown in the figure;

[0058] Figure 2 The mold containing the silk solution in Example 1 is placed on an aluminum rod for directional freezing, and the schematic diagram of the aluminum rod immersed in liquid nitrogen at one end is shown in the figure;

[0059] Figure 3 The SEM images of the hydrogel scaffolds without directional freezing, with directional freezing, and with directional freezing and heating stretching in Example 2 are shown in the figure;

[0060] Figure 4 The stretching curves and mechanical comparison diagrams of the hydrogel scaffolds without directional freezing and with directional freezing in Example 2 are shown in the figure;

[0061] Figure 5 The stress-strain curves obtained by the tensile test of the ND 15 group hydrogel using the mechanical universal testing machine in Example 3 are shown in the figure;

[0062] Figure 6 The stress-strain curves obtained by the fracture toughness test of the ND 15 group hydrogel using the mechanical universal testing machine in Example 3 are shown in the figure;

[0063] Figure 7 The photos of the tensile process of the notched ND 15 hydrogel in Example 3 are shown in the figure;

[0064] Figure 8 The stretching curves and mechanical comparison diagrams of the silk fibroin hydrogels with different concentrations in Example 4 are shown in the figure;

[0065] Figure 9 The DSC diagram of the silk fibroin hydrogel prepared by step c in Example 5 is shown in the figure;

[0066] Figure 10Tensile curve and mechanical comparison of silk fibroin hydrogel obtained by changing heating temperature in step d in Example 5;

[0067] Figure 11 Tensile curve and mechanical comparison of silk fibroin hydrogel obtained by changing stretching ratio in step d in Example 6;

[0068] Figure 12 Tensile stress-strain curve of three groups of hydrogels in Example 7 (left), and the calculated maximum tensile stress and Young's modulus (right);

[0069] Figure 13 HE staining of silk fibroin hydrogel as an external splint for the repair of rabbit Achilles tendon after rupture in Example 8. DETAILED DESCRIPTION

[0070] The preferred embodiments of the present application will be further described in conjunction with the drawings, it should be noted that the following examples are intended to facilitate the understanding of the present application, and do not have any limiting effect on the present application. The raw materials and equipment used in the specific embodiments of the present application are known products, which can be obtained by purchasing commercially available products.

[0071] Example 1 Preparation of silk fibroin hydrogel scaffold provided by the present application

[0072] The preparation method of the silk fibroin hydrogel scaffold provided in this embodiment is prepared according to the process described above, which comprises the following steps: Figure 1 The process described above, which comprises the following steps:

[0073] a. Preparation method of silk solution: boil the domestic silkworm cocoon with 0.02M sodium carbonate for 30min; dry the degummed silk fiber in the fume hood for 12h; dissolve in 9.3M lithium bromide at 60℃ for 4h; dialyze the solution for 72h to remove lithium bromide; air dry the obtained solution in the fume hood for 6h to obtain a silk solution with a concentration of 15%.

[0074] b. Directional freezing: pour the silk solution into a self-made mold with a columnar space of 4*4*20mm. Place the mold above an aluminum rod, which can completely cover the contact surface of the mold, and the contact surface is 4*4mm. One side of the aluminum rod is immersed in liquid nitrogen (as shown in Figure 2 ). After 10min, take out the silk ice block inside and place it in a-80℃ refrigerator overnight.

[0075] c. Ethanol crosslinking: freeze-dry the silk ice block in a freeze-dryer for 72h to obtain a dried scaffold, then soak it in anhydrous ethanol for crosslinking for 6h, and then soak it in water for 24h to ensure complete hydration.

[0076] d. Heating and co-axial stretching: Using a high temperature mechanical testing machine (Model TSE, WANCE), the hydrated hydrogel was clamped between the clamps, and a pre-tension of 2N was applied to the hydrated hydrogel. The material was pre-heated at 95°C for 5 min, and then stretched at a rate of 10 mm / min to 60% of the initial length at 95°C for 15 min. After the stretching, the material was taken out and soaked in water for 24 h to obtain the final hydrogel.

[0077] Example 2 Effect of directional freezing on silk fibroin hydrogel

[0078] In this example, two groups of silk fibroin solutions were prepared. One group was subjected to directional freezing according to the method provided in Example 1, and then cross-linked with ethanol to prepare a directional silk fibroin hydrogel scaffold (DF). The second group was not subjected to directional freezing, and the solution prepared in step a was poured into a mold and directly placed in a -80°C refrigerator. Then, the non-directional silk fibroin hydrogel (ND) was formed by freeze-drying and cross-linking with ethanol. The SEM images of the two groups of hydrogel scaffolds are shown in Figure 3 , wherein Figure 3 b is a parallel arrangement of pipe structures after directional freezing, with parallel pores of 1-10 um, similar to the surface topography of tendons. Figure 3 a is the SEM image of the non-directional hydrogel scaffold, which is significantly different from the directional arrangement of pipe structures of Figure 3 b. The tensile curves of the two groups of silk fibroin hydrogels are shown in Figure 4 , wherein the left graph is the relationship between the stretching ratio and the tensile stress, and the right graph is the comparison between the Young's modulus and the tensile stress. DF 15 in the figure refers to DF prepared from 15% concentration of silk fibroin, and ND 15 in the figure refers to ND prepared from 15% concentration of silk fibroin. According to Figure 4 , it can be clearly seen that the tensile stress and Young's modulus of the directional silk fibroin prepared by directional freezing are significantly improved.

[0079] Example 3 Directional freezing step is a prerequisite for heating and stretching

[0080] This example only involves a silk fibroin hydrogel, which is prepared by pouring the solution prepared in step a into a mold and directly placing it in a -80°C refrigerator (non-directional). Then, the non-directional silk fibroin hydrogel (ND 15 ) is formed by freeze-drying and cross-linking with ethanol. The mechanical properties of the ND 15 group of hydrogels were detected using a mechanical universal testing machine, and the stress-strain curve is shown in Figure 5 . As shown in Figure 5 , ND 15mechanical properties, with the maximum stress of only 0.21 MPa and the fracture strain of about 32%, showing low strength and poor toughness. In order to further test the fracture toughness of the material, pure shear test experiment was carried out on the ND hydrogel. In the test, one pair of ND hydrogels was tested each time, including one original intact hydrogel (Initial ND) and one hydrogel with a pre-made notch (Notched ND), and the stress-strain curve as shown in Figure 6 was obtained by calculating the fracture toughness. Figure 7 The notched ND 15 hydrogel is shown in the photos during the tensile process as shown in Figure 7 The notch of the ND 15 hydrogel rapidly expands during the tensile process and causes the rapid fracture of the hydrogel at a small strain (less than 20%). In combination with the calculated fracture toughness as shown in Figure 6 , the ND 15 hydrogel shows poor fracture toughness. In summary, the ND 15 hydrogel without directional freezing has weak mechanical strength and poor toughness, and will be fractured at a small strain, which is not suitable for subsequent heating and stretching test. In other words, the directional freezing step is a prerequisite for heating and stretching.

[0081] In addition, for the DF 15 prepared by directional freezing, if the directional stretching is not carried out in the direction of directional freezing during the stretching, excellent mechanical properties cannot be obtained, and it is difficult to stretch and easy to tear. Because for the DF 15 prepared by directional freezing, the directionally arranged pipe structure determines that the pipes are easy to separate from each other, if the heating and stretching are not carried out in the direction of directional freezing during the stretching, the DF 15 is easy to tear, and the preparation process of directional freezing combined with heating and stretching cannot be successfully completed.

[0082] Example 4 Effect of Silk Fibroin Concentration on Silk Fibroin Hydrogel

[0083] In this example, four groups of silk fibroin solutions were taken respectively, with concentrations of 5%, 10%, 15%, and 20%, respectively. Each group was subjected to directional freezing according to the method provided in Example 1, and then stretched coaxially at 95°C by 60% to prepare a silk fibroin hydrogel scaffold. The tensile properties of the prepared two groups of hydrogel scaffolds were investigated, and the tensile properties were detected by a biomechanical testing machine. The tensile curves of the silk fibroin hydrogels with different concentrations were obtained as shown in Figure 8 , wherein the left graph is a tensile ratio and tensile stress relationship graph, and the right graph is a Young's modulus and tensile stress comparison graph. DF 15 H 95 S 60is referred to the 15% concentration of silk fibroin hydrogel, which is prepared by heating and stretching at 95℃ with a stretching ratio of 60%. It is prepared by Figure 8 It can be seen that with the increase of silk concentration, the orientation effect of the scaffold is more and more obvious, and the stretching performance of the scaffold is better and better. In theory, the stretching performance of silk fibroin with a concentration of 20% or above will be further enhanced, but due to the limitation of freeze-drying effect, the stretching performance will decrease instead. Therefore, we determine that 15% is a better silk concentration.

[0084] Example 5 Effect of different temperatures of heating and stretching on the performance of silk fibroin hydrogel scaffold

[0085] In this example, the silk fibroin hydrogel is prepared by the method provided in Example 1. First, DSC detection is performed after step c, and the results are shown in Figure 9 In the first heating, with water molecules as plasticizers, the glass transition temperature of silk is about 77℃, and in the second heating, due to the complete loss of water in the scaffold, the plasticizing effect of water molecules is lost, and the glass transition temperature of the silk scaffold is increased to 178℃. Therefore, we set four temperatures in step d, and use different heating temperatures (25℃, 60℃, 95℃, 130℃) for stretching, and investigate the maximum tensile stress of the prepared hydrogel scaffold. The mechanical properties of the scaffold are detected by a biomechanical testing machine, and the tensile curve and mechanical properties of the silk fibroin hydrogel are shown in Figure 10 Before the glass transition temperature (77℃), there is no significant difference in the tensile properties of the hydrogel scaffold prepared at 25℃ and 60℃, and after the temperature rises to the glass transition temperature (77℃), the mechanics of the hydrogel scaffold prepared at 95℃ is significantly higher than that of the other groups, and when the temperature is as high as 130℃, the scaffold is broken when stretched to 60% due to the rapid loss of water molecules, and the mechanical properties decrease instead. This result proves that heating and coaxial stretching at a temperature above the glass transition temperature can significantly enhance the mechanics, and after the loss of water molecules, the glass transition temperature rises, and this effect decreases significantly. Therefore, we determine that 95℃ is a better heating temperature.

[0086] Example 6 Effect of different stretching ratios of heating and stretching on the performance of silk fibroin hydrogel scaffold

[0087] In this example, the silk fibroin hydrogel is prepared by the method provided in Example 1. We set four stretching ratios in step d, and use different stretching ratios (0%, 30%, 60%, 90%) for stretching, and investigate the maximum tensile stress of the prepared hydrogel scaffold. The mechanical properties of the scaffold are detected by a biomechanical testing machine, and the tensile curve and mechanical properties of the silk fibroin hydrogel are shown in Figure 11The mechanical properties of the scaffold gradually increased as the stretching ratio increased from 0% to 60%, indicating that the orientation effect of silk fibroin increased as the stretching ratio increased. However, when the stretching ratio further increased to 90%, the mechanical properties of the scaffold decreased because the high stretching ratio caused partial rupture of the scaffold. Therefore, we determined that 60% was a better stretching ratio.

[0088] Example 7 Directional freezing and heating stretching have a synergistic effect on the improvement of the mechanical properties of the material

[0089] This example relates to three groups of silk fibroin hydrogels, which are prepared by the three preparation methods mentioned in Example 1, Example 2, and Example 4, respectively, including ND 15 , DF 15 , and DF 15 H 95 S 60 hydrogels. Figure 12 The tensile stress-strain curves of the three groups of hydrogels are shown, as well as the maximum tensile stress and Young's modulus obtained by statistics. By comparing ND 15 and DF 15 , it can be concluded that, without the heating stretching step, only directional freezing can increase the maximum stress and Young's modulus of silk fibroin hydrogel by about 6 times and 1 times, respectively. Further heating and stretching of DF 15 hydrogel, as shown in Figure 12 , the maximum stress and Young's modulus of DF 15 H 95 S 60 are increased by 6 times and 5 times compared with DF 15 hydrogel simply subjected to directional freezing, and are increased by 47 times and 15 times compared with ND group.

[0090] In addition, if directional freezing is not performed, and non-directional freezing is not performed, direct heating and stretching are also difficult to stretch and are extremely easy to tear.

[0091] Therefore, directional freezing and heating stretching have a synergistic effect on the improvement of the mechanical properties of non-directional hydrogel.

[0092] Example 8 Silk fibroin hydrogel obtained under optimal conditions for repairing effect on rabbit tendon rupture model

[0093] DF 15 H 95 S 60 hydrogel has similar tensile strength and Young's modulus to tendon, and DF 15 H 95 S 60 hydrogel degrades slowly in vivo and can provide stable mechanics for a long time, and is expected to be used as an artificial tendon substitute. In order to verify DF15 H 95 S 60 To investigate the potential of hydrogel as a tendon patch, we transected the rabbit Achilles tendon and placed DF on the tendon while maintaining tendon tension. 15 H 95 S 60 The hydrogel is sutured to the tendon stump as a lateral splint, providing mechanical compensation and a growth template. Figure 13 In the early stage of repair (4 weeks), all groups showed the appearance of new tendons (blue arrows), but the new tendons in the severance group were disordered, with more and rounder nuclei, more angiogenesis, and partial fat formation (yellow arrows); while in the DF group, 15 H 95 S 60 The new tendons of the DF group were basically arranged in parallel. 15 H 95 S 60 The direction of the nucleus is the same as that of the normal tendon. 15 H 95 S 60 There is only a thin layer of fibrosis and immune reaction around it, which shows that DF 15 H 95 S 60 Good biocompatibility. 15 H 95 S 60 The tendons in the ND group showed little degradation and maintained high integrity (white arrows). Due to the lack of sufficient mechanical conditions, the tendons in the ND group were broken / displaced, and thus could not provide effective mechanical support. The newly formed tendons were similar to those in the severance group. At 12 weeks, the newly formed tendons in the ND and severance groups showed a large wave-like arrangement, while the DF group showed a large wave-like arrangement. 15 H 95 S 60 The new tendons in the DF group became more neat. 15 H 95 S 60 The newly formed tendons in the 3 groups were very close to normal tendons and had no obvious pathological differences from normal tendons.

[0094] Although the present invention is disclosed as above, the present invention is not limited thereto. For example, the present invention can be expanded according to its medical application range. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the claims.

Claims

1. A method for preparing a hydrogel, characterized by, The method comprises the following steps: (1) pouring the silk fibroin solution into a mold to perform directional freezing to obtain a silk fibroin ice block; (2) performing freeze-drying and cross-linking on the silk fibroin ice block, and then soaking the silk fibroin ice block in water to perform hydration to obtain a hydrogel; (3) coaxially heating and stretching the hydrogel. The coaxial heating and stretching in step (3) is performed by heating the silk fibroin hydrogel after hydration to a temperature above the glass transition temperature of the silk fibroin hydrogel and below 100 DEG C, and then performing coaxial stretching, wherein the direction of the coaxial stretching is consistent with the direction of the directional freezing.

2. The production method according to claim 1, wherein The directional freezing in step (1) is performed by placing a freezing source close to the mold, so that the silk fibroin solution in the mold generates ice crystals in a direction away from the contact surface of the freezing source, and the temperature of the freezing source is below -20 DEG C.

3. The production method according to claim 1, wherein Step (1) is: pouring the silk fibroin solution into a cylindrical space in the mold; placing the mold above a metal rod, and immersing one side of the metal rod in liquid nitrogen.

4. The production method according to claim 1, wherein The cross-linking in step (2) is chemical cross-linking, enzyme cross-linking or ethanol cross-linking.

5. The production method according to claim 1, wherein Step (2) is: placing the silk fibroin ice block in a freeze-drying machine to perform freeze-drying, then soaking the silk fibroin ice block in anhydrous ethanol to perform cross-linking, and then soaking the silk fibroin ice block in water to perform hydration.

6. The production method according to claim 5, wherein Step (3) is: preheating the hydrogel after hydration at a temperature of 25-90 DEG C, and then stretching the material by 30-60% of the initial length.

7. Use of the silk fibroin hydrogel prepared by the preparation method of any one of claims 1-6 for preparing a rotator cuff patch or an anterior cruciate ligament substitute.

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