Manufacturing method for high-strength silk fibroin hydrogel artificial tendons / ligaments

Abstract

To provide a method for preparing a silk fibroin hydrogel scaffold having high strength, high biocompatibility, and biological microstructure.SOLUTION: There is provided a method for preparing a silk fibroin hydrogel scaffold having high strength, high biocompatibility, and biological microstructure for repair and reconstruction of tendons / ligaments by utilizing a natural silk fibroin material through directional freezing and coaxial hot stretching. The heating temperature must be higher than a glass transition temperature of the silk fibroin material, such that a maximum tensile strength of the silk fibroin hydrogel scaffold increases, a microscopic directional pore structure is formed, and finally a mechanically reinforced hydrogel scaffold with good biocompatibility is obtained.SELECTED DRAWING: Figure 12A

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 2023112411598, filed on 22 September 2023, and the entire contents of that application, including but not limited to the specification, abstract, claims and attached drawings, are incorporated into this application by reference.

[0002] The present invention relates to the technology of hydrogel preparation, and more specifically to a method for preparing high-strength silk fibroin hydrogels, in particular a method for preparing high-strength silk fibroin hydrogels that can be used as substitutes for rotator cuffs, anterior / posterior cruciate ligaments, medial retinaculum, etc. [Background technology]

[0003] Tendon / ligament injuries are one of the most common sports injuries. Human tendons and ligaments are mainly composed of high-density type I collagen, with a low cell content and weak angiogenic capacity, making them difficult to heal naturally once damaged. Patients with large tendon / ligament injuries, such as massive rotator cuff tears or anterior cruciate ligament tears, often require patch-assisted treatment. Currently, there are many methods for preparing tendon / ligament repair materials, but conventional techniques such as autologous and allogeneic materials have insufficient sources, making them prone to problems such as complications at the donor site, rejection, and disease transmission. Currently, a few artificial materials, such as France's "LARS" ligament and China's "Ligetai," are used clinically, but all of them have problems such as low tensile strength, poor biocompatibility, and difficulty in fusing with host tissue, making them prone to loosening of bone tunnels and stress fractures. These drawbacks lead to poor repair outcomes for tendon / ligament injuries, making the discovery of scaffold materials with both excellent mechanical properties and biocompatibility a hot spot in research.

[0004] Silk fibroin is widely used in the manufacture of tissue engineering scaffolds and possesses good biocompatibility, biodegradability, and excellent mechanical properties. Silk fibroin is readily available and can be extracted from raw silk. Raw silk has a unique protein macromolecular structure, with silk fibroin as the core and sericin attached to the surface. Silk fibroin is obtained by removing the rubbery substance from raw silk. Silk fibroin contains 18 amino acids, most of which are glycine, alanine, and serine. Silk fibroin is completely broken down in the body, and during this breakdown process, the cellular microenvironment becomes rich in numerous amino acids, especially proline and lysine, which are the main raw materials for collagen synthesis. The repeating GAGAG sequence in silk fibroin can self-assemble to form a secondary β-sheet structure, which gives silk fibroin excellent mechanical strength and toughness. Reports indicate that the toughness of silk fibroin is superior to that of the best synthetic materials. At the same time, silk fibroin possesses tunable mechanical properties. By adjusting the proportion of β-sheet structures in silk fibroin, the mechanical strength of silk fibroin material can be further improved, surpassing currently most commonly used biodegradable polymer materials such as collagen and polylactic acid. Furthermore, the surface of raw silk material is easily processed and modified, allowing it to be formed into various shapes, from liquid solutions to organic solid states such as gels, pipes, hydrogels, microspheres, and films. These properties of raw silk material make it promising for tissue engineering applications.

[0005] However, conventional silk fibroin still struggles to achieve and maintain mechanical strength comparable to that of human tendons / ligaments. Therefore, there is an urgent need to develop novel silk fibroin scaffolds with superior initial mechanical properties and long-term durability. [Overview of the project]

[0006] To solve the above problems, the present invention provides a method for preparing a high-strength silk fibroin hydrogel scaffold for tendon / ligament, which utilizes a natural silk fibroin material and realizes an improvement in the maximum tensile strength of the silk fibroin hydrogel scaffold by a processing method of directional freezing and heating coaxial tension (higher than the glass transition temperature of silk fibroin). It has a fine directional pore structure, and as a result, it has good biocompatibility and obtains a mechanically strengthened hydrogel scaffold, which can be used for the manufacture of tendon / ligament scaffolds and can achieve the same mechanical strength as human tendons / ligaments (e.g., tendon plates, cruciate ligaments, Achilles tendons). In addition, it has excellent biocompatibility and degradability, and its fine morphology has micron-scale directional pores, which is helpful for the directional growth of cells on the surface and inside of the material, and can achieve a higher repair efficiency.

[0007] Therefore, according to one aspect of the present invention, there is provided a method for preparing a hydrogel, the method comprising:

[0008] performing directional freezing on a silk fibroin solution to change the solvent in the solution from a liquid state to a solid state (step (1));

[0009] sublimating and drying the solid-state solvent in the product obtained in step (1) (step (2));

[0010] heating and stretching the product after freeze-drying to obtain the above hydrogel (step (3)).

[0011] In some embodiments, there are various methods for sublimation in step 2, but any method that can directly change the solvent from a solid state to a gaseous state can be used, and in this method, the sublimation method can be selected according to the properties of the solvent, for example, freeze-drying is commonly used. If the solvent is water, the solidified ice can be directly placed in a freeze-dryer and freeze-dried, thereby causing the water to evaporate or sublimate directly from the solid ice, leaving the final silk fibroin. In some embodiments, the solvent is completely evaporated, where sublimation can be 100% evaporation, or 99% to 80% of the solvent can sublimate directly from a solid state to a gaseous state.

[0012] In some embodiments, the solvent for the silk fibroin solution includes water, such as deionized water, pure water, mineral water, or any water molecules found in nature. Of course, the solvent does not have to be water; any other solvent that can dissolve silk fibroin and changes to a solid state at low temperatures can be used.

[0013] In some embodiments, the silk fibroin solution of the present invention is obtained by removing the rubbery substance from natural raw silk. It should be understood that in some embodiments, the silk fibroin obtained by removing the rubbery substance from raw silk can be purchased directly. In some embodiments, artificially synthesized silk fibroin can also be used.

[0014] In this invention, "directional freezing" means cooling a polymer solution by placing it in a temperature field. As the temperature decreases, the solvent (usually water) solidifies in a gradient in the direction of the gradual decrease in temperature. The ice crystal columns formed by this gradual solidification push out the polymer chains, rearranging them directionally and embedding them between the ice crystal columns. Generally, a cylindrical or rectangular container (cavity) is placed in or in contact with a cold source, and the cavity is filled with a silk fibroin solution. The solution gradually solidifies in the direction away from the cold source from the point of contact, and ice crystal columns are gradually formed along this direction. That is, during freezing, the solvent in the solution gradually freezes and solidifies, gradually changing from a liquid state to a solid state, and ice crystal columns (when the solvent is water) are formed along the direction of gradual freezing. In this process, the ice crystals, acting as orientation templates for the polymer chains, function as physical traps and play a role in directionality. The term "gradually" as used in this invention is a relative concept. When a certain volume of solution comes into contact with a cold source, the solution always begins to change from a liquid state to a solid state near the cold source first. Then, as time passes, the solution solidifies and continues to move away from the cold source until the entire solution becomes solid. During this process, the structure of ice crystals is also gradually formed, and as time passes, the structure of the ice crystals also gradually elongates, with the direction of elongation being substantially the same as the direction of solidification.

[0015] The term "directional freezing" as used herein can also be understood as follows: When a solution is brought close to the temperature at which the solvent solidifies (when there is one solidification temperature source), the solvent in the solution gradually solidifies from the direction closer to the solidification temperature source toward the direction away from the solidification temperature source, thereby achieving a gradual solidification from a liquid state to a solid state. Such solidification is directional; when the solidification temperature is from a single temperature source, the direction of solidification is unidirectional; when the solution is between two solidification temperature sources, the solvent in the solution gradually solidifies from both ends toward the center, and the direction of solidification is bidirectional. Indeed, when the solidification temperature source is in the center of the solvent, the solvent gradually solidifies from the center toward the outside, and the direction of solidification is a dispersion from the center of the solvent toward both ends. Whether unidirectional, two opposing directions, or divergent directions, these are all specific methods of implementing directional freezing and solidification. In some embodiments, the directional freezing described above involves changing the solvent dissolving silk fibroin from a liquid state to a solid state, for example, changing water from a liquid state to a solid state, for example, into the state of ice.

[0016] Naturally, directional freezing does not include such methods, except here, the method of placing the entire solution containing silk fibroin at the bottom of a refrigerator and placing it in a three-dimensional freezing environment where the ambient temperature is lower than the solidification temperature of the solution solvent, which can change the solvent from a liquid state to a solid state, is not what is meant by "directional freezing" as described in this invention.

[0017] Therefore, directional freezing can form heterogeneous structures in various directions, allowing materials to be aligned in various directions within space. The mechanical properties in the orientation direction using directional freezing are far stronger than those without directional freezing. Furthermore, the spatial occupancy effect of ice crystals or the solvent solid phase increases the local degree of aggregation of silk fibroin molecules, further improving the mechanical properties. Compared to non-directionally frozen silk fibroin hydrogels, the maximum tensile stress of directionally frozen silk fibroin hydrogels can be increased several times. Moreover, directional freezing technology allows silk fibroin solutions to form parallel-aligned pipe structures with parallel pores of 5-10 μm, similar to the microstructure of tendons. This microstructure can, on the one hand, guide and stimulate tendon stem cells to differentiate into tendon cells, and on the other hand, promote the penetration, growth, and directional alignment of cells into the scaffold, thereby promoting the efficiency and effectiveness of tendon regeneration.

[0018] In some embodiments, to facilitate specific directional freezing, a solution containing silk fibroin can be placed in a container, and then directional freezing can be performed on the solution in the container. For example, the container can be of any shape, such as a sphere, cube, rectangular prism, or other container shape. In some embodiments, the internal space of the container is a rectangular prism, and for example, the cross-section of the rectangular prism may be any shape such as a circle, rhombus, square, or ellipse. For example, the container is a rectangular prism, and when a solution is poured or placed in such a container, the solution is cooled from one end by the freezing source, gradually freezing and solidifying from one end to the other, gradually freezing along the longitudinal direction of the container, the solvent changes from a liquid state to a solid state along the longitudinal direction, and as a result, ice forms.

[0019] In some embodiments, step (3) involves directional tensioning of the material. In some embodiments, the direction of the directional tensioning is the same as the direction of directional freezing. For example, if a silk fibroin solution freezes and solidifies in one direction, the tensioning direction is also a single tensioning direction and is substantially the same as the freezing direction. It should be understood that directional freezing is in one direction (direction of a single arrow) from one end to the other, while tensioning can be performed outward from two points (direction of a double arrow), although the tensioning directions are not the same, they are all on the same line. For example, directional freezing is the gradual freezing and solidification of a rectangular container from one end to the other along the longitudinal direction, while tensioning may be performed along the longitudinal axis, in two directions, or along the longitudinal axis.

[0020] In some embodiments, the silk fibroin material is heated to a certain temperature during tensioning. In some embodiments, the heating temperature is higher than the glass transition temperature of the silk fibroin material, and the mechanical properties of the material can be significantly improved by heated coaxial or directional tensioning. Both coaxial tension with directional freezing and downward tension with directional freezing direction impart a specific strain to the material, allowing the material to be stretched to a specific length. The resulting tensile stress rearranges the molecules within the material, thereby giving the material a specific orientation in the tensile direction, further improving the tensile strength in that orientation.

[0021] Amorphous polymers have three mechanical states: glassy state, highly elastic state, and viscous flow state. At low temperatures, the material is in a hard solid state similar to glass, and even when external force is applied, only very small deformation occurs. This is the glassy state. As the temperature continues to rise within a certain range, the amount of deformation of the material increases significantly, and within that temperature range, the amount of deformation becomes relatively stable. This is the highly elastic state. The glass transition temperature (Tg) refers to the temperature at which the transition from the glassy state to the highly elastic state occurs. Silk fibroin is in the glassy state at room temperature or room temperature (25 degrees Celsius), and only slight deformation occurs even under coaxial tension. When heated, silk fibroin exceeds its Tg, entering a highly elastic state. In this state, the movement of silk fibroin molecules becomes more active, the range of motion expands, and the silk fibroin material in this state is easily deformed, allowing it to be pulled to higher magnifications during coaxial or directional tension. The intensification of molecular motion helps in the formation of orientation at the molecular level.

[0022] Regarding tensile equipment, tensile testing can be automatically completed using conventional equipment, such as high-temperature mechanical testing machines. High-temperature mechanical testing machines are scientific instruments used in the field of materials science and mainly include an extensometer, a high-temperature furnace for heating, and bar and plate clamps for fixing the tensile material, such as the silk fibroin and directional frozen silk fibroin material of the present invention. For example, all the equipment introduced on this site can complete the tensile testing of the present invention. http: / / www.cxwannengsyj.com / html / 2019 / gaodiwen_0521 / 1596.html?sdclkid=ALos152NxSDibL-pA52G&bd_vid=10212208613159825381. , https: / / www.zhongguoqingji.com / 685cc91a-bdab-2768-53cf-5bde625d2d7f / GDWWNSYJ.shtml?bd_vid=12561170255290571953,

[0023] The glass transition temperature of dry silk fibroin film is approximately 178°C. When water molecules are used as a plasticizer, the conditions used in this invention can lower the glass transition temperature to 77°C, thereby significantly reducing the difficulty of operation and lowering costs.

[0024] Accordingly, in some embodiments of the present invention, directional freeze-dried silk fibroin is freeze-dried, the solidified solvent is evaporated to leave only the silk fibroin itself, then it is crosslinked and / or hydrated, and then directional tensile is performed. Thus, because it can be carried out at a low temperature, the difficulty of operation is reduced and costs are lowered. The present invention can increase the maximum tensile stress of silk fibroin hydrogel by about 10 times by performing coaxial tensile on hydrated silk fibroin at different temperatures above Tg and different tensile strains. The directional freeze-drying and heat tensile used in the present invention also produce a remarkable synergistic effect, thereby increasing the maximum tensile strength of silk fibroin hydrogel by almost 100 times, and it also exhibits excellent biocompatibility. The fine directional pore structure is useful for cell growth and further aids in the repair of tendon injuries.

[0025] Heat tensile strength must be performed after directional freezing, and coaxial tensile strength must be performed in the direction of directional freezing. 1. The directional freezing step is a prerequisite for heat tensile strength, but if tensile strength is applied directly without directional freezing, the silk fibroin hydrogel will be directly fractured. 2. If coaxial tensile strength is not applied in the direction of directional freezing after directional freezing, the silk fibroin hydrogel will also be directly fractured.

[0026] Furthermore, the above preparation method is

[0027] Step (1) involves injecting a silk fibroin solution into a mold and performing directional freezing to obtain a silk fibroin ice block,

[0028] Step (2) involves freeze-drying silk fibroin ice blocks to crosslink them, then immersing them in water to hydrate them and obtain a hydrogel,

[0029] The process includes (3) a step of applying heat tension to the hydrogel.

[0030] In some embodiments, the direction of heat tensile force coincides with the direction of directional freezing.

[0031] Furthermore, the directional freezing described in step (1) refers to the process in which, as the freezing source is brought closer to the mold, the silk fibroin solution in the mold begins to approach the contact surface of the freezing source and then directionally generates ice crystals in the direction away from the contact surface of the freezing source. In some embodiments, the temperature of the freezing source is below -20°C, -10°C, -5°C, -2°C, or below -1°C.

[0032] Any device or product that has the effect of lowering the temperature may be used as a freezing source in the present invention, or in any case, any freezing source that solidifies the solvent in a solution containing silk fibroin is acceptable. For example, different freezing sources can be set depending on the freezing point temperature of the solvent. The "freezing source" set in the present invention depends on the solvent in which the silk fibroin is dissolved, as well as on environmental conditions, and if the solvent and environment are different, the freezing and solidification temperature of the solvent will also be different. For example, if the solvent is pure water, as atmospheric pressure increases, the freezing point of water decreases, and the solidification temperature of water becomes below 0°C. Naturally, the lower the temperature, the shorter the solidification time of water. The solution has a certain height (for example, the height of the solution is 1 mm to 200 cm), and the time it takes for the entire solution to solidify also differs. For example, if the solvent is water and the thickness is 1 mm, solidification can be achieved below 0°C, but if the thickness is 10 cm or 20 cm, a lower temperature and a longer time may be required. It should be understood that those skilled in the art can read the contents of the present invention and make arbitrary selections according to the actual situation, and ultimately, it is acceptable as long as the solution containing silk fibroin can solidify.

[0033] In some embodiments, the freezing source can be realized using liquid nitrogen. In some embodiments, when performing directional freezing, directional freezing of the silk fibroin solution can be achieved by connecting one end of a metal element to liquid nitrogen and the other end to one end of a container containing the silk fibroin solution. In some embodiments, step (1) is to place the silk fibroin solution in the cylindrical space of a mold, place the mold above a metal rod, and immerse one end of the metal rod in liquid nitrogen.

[0034] In some embodiments, a metal rod acting as a freezing source is located below the mold and in full contact with the bottom surface of the mold. In the event of freezing, ice crystals of the silk fibroin material grow upward from the lower cold source, with a certain directionality, which is perpendicular to the bottom surface in contact with the freezing source, and then grows away from the freezing source.

[0035] In some embodiments, the metal rod serving as the freezing source is located on the side of the mold and in full contact with the side of the mold. In the event of freezing, ice crystals in the silk fibroin material begin to grow directionally from the side in contact with the freezing source, with the direction of ice crystal growth being perpendicular to the bottom surface of the freezing source, and then growing away from the freezing source.

[0036] In some embodiments, a metal rod serving as a freezing source is located on the top surface of the mold and is in full contact with the top surface of the mold. In the event of freezing, ice crystals of the silk fibroin material begin to grow downward from the top surface in contact with the freezing source, with the direction of ice crystal growth being perpendicular to the bottom surface of the freezing source, and then growing away from the freezing source.

[0037] In some embodiments, step (1) is to place a silk fibroin solution into a mold having a cylindrical space of 4*4*20 mm. The mold is placed above an aluminum rod having a width that completely covers the contact surface with the mold, and one side of the aluminum rod is immersed in liquid nitrogen. After 10 minutes, the mold is removed, the silk fibroin ice block inside is taken out and placed in a refrigerator at -80°C overnight.

[0038] Furthermore, after directional freezing and drying, the silk fibroin is crosslinked. Therefore, in some embodiments, for example, the crosslinking described in step (2) is chemical crosslinking or enzymatic crosslinking.

[0039] Since silk fibroin is water-soluble, crosslinking is necessary to form a hydrogel. On the one hand, crosslinking can stabilize the silk fibroin scaffold in aqueous solution, and on the other hand, it can further improve the strength of the material.

[0040] One important point to note is that crosslinking should be performed after directional freezing is complete. If the molecular structure is fixed by crosslinking beforehand, the ice crystals will not be able to align directionally as they grow during directional freezing, preventing the formation of fine directional pores, and ultimately causing the scaffold to lose its directional structure.

[0041] The present invention can utilize any crosslinking method, such as ethanol crosslinking, other chemical crosslinking, or enzymatic crosslinking, and as long as the silk fibroin material can be crosslinked, it can be used in the present invention and is within the scope of protection of the present invention.

[0042] Furthermore, step (2) involves freeze-drying the silk fibroin ice block in a freeze-dryer, crosslinking it by immersing it in anhydrous ethanol, and then hydrating it completely by immersing it in water.

[0043] In some embodiments of the present invention, anhydrous ethanol is used for crosslinking. However, it is necessary to freeze-dry the silk fibroin ice block before crosslinking. When water is used as the solvent, the ice block is evaporated and removed while retaining the directional silk fibroin. This is because if the silk fibroin ice is directly crosslinked without freeze-drying, and the low-temperature silk fibroin ice block is brought into contact with relatively high-temperature ethanol, the ice crystals will melt before ethanol crosslinking, causing the internal microstructure to break down. After freeze-drying the silk fibroin ice block, a complete silk fibroin scaffold with a fine directional structure is obtained. Subsequently, this is placed in anhydrous ethanol to undergo intermolecular crosslinking, forming a more stable silk fibroin hydrogel structure.

[0044] In some embodiments, the material can be hydrated by immersion in water after crosslinking, for the following reasons: The glass transition temperature of dry silk fibroin material is approximately 178°C. By using water molecules as a plasticizer with changes in humidity, the glass transition temperature can be lowered to below 77°C. After lowering the glass transition temperature, the difficulty of heat tensile strength is significantly reduced, costs are lowered, and coaxial tensile strength is reduced. Furthermore, if coaxial tensile strength is performed at temperatures above 100°C, the water in the hydrogel evaporates rapidly, resulting in the loss of water molecules as a plasticizer, making the hydrogel brittle and limiting the tensile length. Therefore, hydrated silk fibroin can be achieved at temperatures below 100°C, such as 95°C, 85°C, 75°C, 65°C, 55°C, 45°C, 35°C, 25°C, or below 15°C, preferably below 77°C. Sufficient hydration refers to immersion in water to the extent that the quality of the scaffold does not fundamentally change, indicating that the hydration is sufficient, and is generally immersed for 48 hours or more.

[0045] In some embodiments, the silk fibroin ice block can be placed in a freeze-dryer and freeze-dried for 72 hours to obtain a dry scaffold, then immersed in anhydrous ethanol for 6 hours to crosslink, and then immersed in water for 48 hours to fully hydrate.

[0046] Furthermore, the heating and tensioning described in step (3) refers to performing coaxial tensioning after heating the silk fibroin to a temperature above its glass transition temperature, and the direction of the coaxial tensioning coincides with the direction of directional freezing.

[0047] When the direction of coaxial tension coincides with the direction of directional freezing, the resulting scaffold has superior mechanical properties, possesses fine directional pores, and is conducive to cell growth.

[0048] After directional freezing, drying, and crosslinking, the hydrogel exhibits longitudinal stripes, and the direction of the fine pores formed by directional freezing is visible to the naked eye. In this case, heating above the glass transition temperature of silk fibroin and applying directional coaxial tension in the same direction rapidly improves mechanical strength.

[0049] Furthermore, step (3) involves first preheating the hydrated hydrogel to a temperature of 25-130°C, and then stretching the material to 30-90% of its initial length.

[0050] In some embodiments, the heating temperature only needs to be above the glass transition temperature of silk fibroin, and the glass transition temperature of the hydrated silk fibroin hydrogel can be lowered to below 77°C, for example, to 25°C.

[0051] Furthermore, step (3) involves first preheating the hydrated hydrogel to a temperature of 25-130°C, then stretching the material to 30-90% of its initial length, holding it for 15 minutes, then removing it and immersing it in water to hydrate it, thereby obtaining the final hydrogel.

[0052] In some embodiments, heating and tensile strength can be completed using a heating and tensile strength device such as a high-temperature mechanical testing machine, provided that heating and tensile strength can be performed simultaneously.

[0053] In some embodiments, the pre-tensile force for heat tensile only needs to be sufficient to tensile the hydrogel so that it does not shrink significantly due to the pre-tensile force.

[0054] In some embodiments, the heat tensile strength is not constant, and the mechanical properties of the hydrogel can be improved as long as it is tensed, so it can be tensed according to the actual needs, as long as the scaffold does not break. If tensed to more than 90% of the initial length, the material may partially break.

[0055] In some embodiments, step (3) involves applying a pre-tensile force of 2N to the hydrated hydrogel, preheating it at 95°C for 5 minutes, then stretching the material at a rate of 10 mm / min at 95°C to 30-90% of its initial length, holding it for 15 minutes, after which it is removed and immersed in water to hydrate it to obtain the final hydrogel.

[0056] Furthermore, the method for preparing the silk fibroin solution described in step (1) is as follows: After removing the silkworm pupae from the raw silk, they are placed in a 0.02 M sodium carbonate solution and boiled for 30 minutes to remove the rubbery substance. The silk fibroin fibers after rubber removal are dried in a fume hood for 12 hours. Then, they are dissolved in 9.3 M lithium bromide at 60°C for 4 hours. The solution is dialyzed using a dialysis bag for 72 hours to remove the lithium bromide. The resulting solution is air-dried in a fume hood for 6 hours to obtain a 15% silk fibroin solution.

[0057] This invention uses natural raw silk, removes the rubbery substance from the raw silk, prepares a silk fibroin solution, freeze-dries the silk fibroin solution using directional freezing, crosslinks it with anhydrous ethanol, thoroughly hydrates the resulting scaffold, then stretches 60% of its initial length at a speed of 10 mm / min under high temperature conditions above the glass transition temperature (95°C), holds for 15 minutes, removes the scaffold, and hydrates it to obtain the final hydrogel.

[0058] In another embodiment, the present invention provides an application for a method of improving the mechanical properties of a material, the method of first performing directional freezing on the material and freeze-drying it, and then performing coaxial heating tension, wherein the heating temperature is above the glass transition temperature of the material, and the material is PVA, gelatin, etc.

[0059] This invention provides a collaborative processing method combining heat tensile and directional freezing. Here, directional freezing not only imparts a fine directional pore structure to the material, but can also improve the mechanical properties of the material through the formation of this anisotropic structure. Furthermore, if the material is subjected to further heat tensile above its glass transition temperature after directional freezing, the mechanical properties of the material can be further significantly improved, and the maximum tensile stress of the material can be increased. At the same time, the micron-scale directional pore structure of the hydrogel guides and stimulates cell differentiation through microtopological topography, on the one hand, and facilitates cell penetration on the scaffold, thereby promoting tissue repair.

[0060] The cooperative processing method of directional freezing and heat tensile provided by the present invention is suitable not only for silk fibroin materials but also for improving the mechanical properties of other materials.

[0061] Furthermore, in another embodiment, the present invention provides an application for a silk fibroin hydrogel for manufacturing a rotator cuff patch or anterior cruciate ligament substitute, the silk fibroin hydrogel being obtained by directional freezing of a silk fibroin solution, freeze-drying, and then heat-tensile, wherein the heat temperature is above the glass transition temperature of silk fibroin.

[0062] In short, the present invention provides for a tensile (called heated tensile) processing method above the glass transition temperature, and a cooperative processing method of heated tensile and directional freezing, which is used to improve the mechanical properties of materials, obtain surface microtopography, and help improve tendon repair efficiency.

[0063] The silk fibroin hydrogel scaffold provided by the present invention has the following beneficial effects.

[0064] 1. Excellent mechanical properties; while the maximum stress of conventional silk fibroin hydrogels is approximately 0.02-0.2 MPa, the present invention can reach over 10 MPa, which is several hundred times greater.

[0065] 2. The directional topology of the hydrogel surface promotes the directional arrangement of tendon stem cells.

[0066] 3. Excellent biocompatibility; the raw silk material itself possesses excellent biocompatibility, and since no organic reagents that are difficult to remove are used in the preparation process of the hydrogel scaffold, the resulting scaffold has high biocompatibility.

[0067] 4. Raw silk material is easier to obtain than autologous tendons, and the preparation process is also simpler.

[0068] 5. The provided directional freezing and heat tensile cooperative processing method is suitable not only for silk fibroin materials but also for improving the mechanical properties of other materials. [Brief explanation of the drawing]

[0069] [Figure 1A] This is a flowchart for the preparation of a silk fibroin hydrogel scaffold. Figure 1A is a schematic diagram illustrating the principle of directional freezing, where the conical structure in the cube is a schematic diagram of the ice crystal structure, the black dots are water molecules, and the curves between water molecules are a schematic diagram of silk fibroin. [Figure 1B] Figure 1B is a schematic diagram illustrating the principles of bridge bridging and hydration. [Figure 1C] Figure 1C is a schematic diagram illustrating the principle of directional tension. [Figure 2A] In a specific embodiment of the present invention (Example 1), the diagram shows a schematic operation in which a mold containing a silk fibroin solution is placed on an aluminum rod for directional freezing, and one end of the aluminum rod is immersed in liquid nitrogen. [Figure 2B] This is a schematic diagram of the principle and operating structure of directional freezing in a specific embodiment of the present invention. [Figure 3] These are SEM images of the hydrogel scaffolds heated and stretched in Example 2, before, during, and after directional freezing. As can be seen, the hydrogel without directional freezing exhibits an irregular pore structure at a fine level, while the hydrogel with directional freezing exhibits a directional pore structure at a fine level. [Figure 4A] This figure shows a comparison of tensile curves in Example 2, both without and with directional freezing. [Figure 4B] This diagram shows the mechanical comparison results in Example 2, with and without directional freezing. [Figure 5] This is the stress-strain curve obtained by performing a tensile test on ND15 hydrogel using a mechanical universal testing machine in Example 3. [Figure 6] This is the stress-strain curve obtained by performing a fracture toughness test on an ND15 hydrogel using a mechanical universal testing machine in Example 3. [Figure 7] These are photographs of the notched ND15 hydrogel during the tensile process in Example 3 (the leftmost image shows the start of tensile stress, the middle image shows fracture during the tensile process, and the rightmost image shows the result after complete fracture during the tensile process). [Figure 8A] This is a comparison chart of the tensile curves of silk fibroin hydrogels with different concentrations in Example 4. [Figure 8B] This diagram shows the mechanical comparison results of silk fibroin hydrogels with different concentrations in Example 4. [Figure 9] This is a DSC diagram of the silk fibroin hydrogel prepared in step c of Example 5. [Figure 10A] This is a comparison diagram of the tensile curves of silk fibroin hydrogels obtained by changing the heating temperature in step d of Example 5. [Figure 10B] This diagram shows the mechanical comparison results of silk fibroin hydrogels obtained by changing the heating temperature in step d of Example 5. [Figure 11A] This is a comparison diagram of the tensile curves of silk fibroin hydrogels obtained by changing the tensile magnification in step d of Example 6. [Figure 11B] This diagram shows the mechanical comparison results of silk fibroin hydrogels obtained by changing the tensile strength in step d of Example 6. [Figure 12A]Figure 12A shows the tensile stress-strain curves of the three groups of hydrogels in Example 7, and Figure 12B shows the calculated maximum tensile stress and Young's modulus. [Figure 12B] Figure 12A shows the tensile stress-strain curves of the three groups of hydrogels in Example 7, and Figure 12B shows the calculated maximum tensile stress and Young's modulus. [Figure 13] This is an HE-stained image of silk fibroin hydrogel used as a splint to repair a ruptured rabbit Achilles tendon in Example 8. [Modes for carrying out the invention]

[0070] Preferred embodiments of the present invention will be described in more detail below with reference to the drawings. It should be noted that the embodiments described below are intended to facilitate understanding of the present invention without limiting it. The raw materials and equipment used in the specific embodiments of the present invention are all known products and are available by purchasing commercially available products.

[0071] Example 1 Preparation of a silk fibroin hydrogel scaffold provided by the present invention The method for preparing the silk fibroin hydrogel scaffold provided by this embodiment is carried out according to the flow shown in Figure 1, and the method includes the following steps.

[0072] a. In the method for preparing the silk fibroin solution, silkworm pupae (5g) are boiled in a 0.02M sodium carbonate solution (2L) for 30 minutes, the silk fibroin fibers after removing the rubbery substance are dried in a fume hood for 12 hours, then dissolved in 9.3M lithium bromide at 60°C for 4 hours, the solution is dialyzed using a dialysis bag for 72 hours to remove the lithium bromide, and the resulting solution is air-dried in a fume hood for 6 hours to obtain a 15% concentration aqueous solution of silk fibroin.

[0073] b. In directional freezing, a silk fibroin solution is placed in a cylindrical space of a handmade mold. The mold has a cylindrical space measuring 4*4*20 mm (cubic opening, size 4*4 mm, depth 20 mm), and this space contains an aqueous silk fibroin solution. Typically, this space is filled with the solution, and the mold material can be any metal such as aluminum, iron, or alloy. In this embodiment, the abrasive material is an aluminum alloy. Naturally, in other examples, any space such as a cylinder, cube, or cuboid is acceptable, and all of these spaces have openings for freezing, into which the silk fibroin solution can be placed to achieve directional freezing.

[0074] One end of the aluminum rod is immersed in liquid nitrogen (Figure 2A), and in this way the aluminum rod is cooled by the liquid nitrogen and its temperature drops. A mold containing the silk fibroin solution is placed on the other end (the bottom of the cylindrical space is in contact with the liquid), and the 4*4 mm bottom of the mold is in complete contact with the surface of the aluminum rod. Due to the heat transfer effect of the aluminum rod, directional freezing occurs gradually upward from the bottom of the cylindrical space, and the solution gradually solidifies from the bottom, especially the water which gradually solidifies upward from the bottom, and the formed ice crystals also gradually spread from the bottom towards the opening (direction of the arrow in step 1 in Figure 1A). After 10 minutes, the mold is removed, the silk fibroin ice block inside is taken out and placed in a refrigerator at -80°C overnight.

[0075] For example, referring to Figure 2B, the opening 10 of the cylindrical space 12 is at the top, in which case one end 13 of the aluminum rod may be located at the bottom 11, or the bottom 11 of the cylindrical space may be located on the surface of the aluminum rod, and the other end 14 of the aluminum rod is in contact with liquid nitrogen. In this way, the solution in the cylindrical space gradually solidifies from the bottom 11 toward the opening 10, and as a result, the formed ice crystals also spread toward the opening, and directional freezing occurs in the direction of the arrow in Figure 2(b).

[0076] c. In ethanol crosslinking, the silk fibroin ice block is placed in a freeze-dryer and freeze-dried for 72 hours, allowing the ice block to evaporate and volatilize to obtain a dry scaffold. Next, the silk fibroin scaffold is immersed in anhydrous ethanol for 6 hours to crosslink, and then immersed in water for 24 hours to completely hydrate it.

[0077] d. In heated coaxial tensile testing, a high-temperature mechanical testing machine (TSE type, WANCE, model number: ETM105D) is used. The hydrogel hydrated in step C above is clamped, a pre-tensile force of 2N is applied to the hydrated hydrogel, and it is preheated to 95°C for 5 minutes. Then, at a temperature of 95°C, the material is tensed at a speed of 10 mm / min to 60% of its initial length, held for 15 minutes, the temperature is increased from the surroundings towards the clamp (heat source), and then it is removed and hydrated in water for 24 hours to obtain the final hydrogel. The tensile direction is the same as that of the cylindrical space for freezing (the direction of the arrow in Figure 1C is the longitudinal tension to both ends), that is, the tensile direction is along the longitudinal upward direction to a depth of 20 mm, which can be understood as longitudinal tension.

[0078] Example 2: Effect of directed freezing on silk fibroin hydrogel In this example, two groups of silk fibroin solutions were collected. One group was subjected to directional freezing according to the method provided in Example 1, and then crosslinked with ethanol to prepare a directional silk fibroin hydrogel scaffold (DF). For the second group, directional freezing was not performed. The solution prepared in step a was placed in a mold, placed directly into a refrigerator at -80°C, freeze-dried, and then crosslinked with ethanol and hydrated to form a non-directional silk fibroin hydrogel (ND). SEM images of the two prepared hydrogel scaffolds are shown in Figure 3. Figure 3b (center) shows a parallel-aligned pipe structure formed after directional freezing, with parallel pores of 1-10 μm, similar to the surface topology structure of tendons. Figure 3a is an SEM image of the non-directional hydrogel scaffold (far left), which is significantly different from the directional-aligned pipe structure in Figure 3b. The tensile curves of the two groups of silk fibroin hydrogels are shown in Figures 4A and 4B. Figure 4A is a diagram showing the relationship between tensile strength and tensile stress, and Figure 4B is a comparison diagram of Young's modulus and tensile stress. Here, DF 15 This refers to DF prepared with 15% silk fibroin, and ND. 15 This refers to ND prepared with 15% silk fibroin. As is clear from Figure 4, the tensile stress and Young's modulus of silk fibroin prepared by directional freezing are significantly improved, with the tensile stress increasing by approximately 6 times and the Young's modulus by approximately 1 time.

[0079] Example 3 Directional freezing step as a prerequisite for heat stretching This embodiment relates only to silk fibroin hydrogel, and its preparation process involves placing the solution prepared in step a into a mold, placing it directly into a -80°C refrigerator (non-directional), then freeze-drying and crosslinking with ethanol to produce a non-directional silk fibroin hydrogel (ND 15 ) is formed using a mechanical universal testing machine. 15 The mechanical properties of the hydrogels in the group were tested, and the stress-strain curves shown in Figure 5 were obtained through tensile testing. As shown in Figure 5, ND 15Its mechanical properties are poor, with a maximum stress of only 0.21 MPa, a fracture strain of about 32%, low strength, and poor toughness. To further test the fracture toughness of the material, a pure shear test experiment is conducted on ND hydrogel. In this test, one pair of ND hydrogels is tested each time, which includes the original complete hydrogel (Initial ND) and the hydrogel with a notch created (Notched ND). A tensile test is performed on each pair of hydrogels to obtain the stress-strain curve shown in Figure 6, and the fracture toughness is obtained by calculation. Figure 7 shows the photo of the notched ND 15 hydrogel. As shown in Figure 7, the notch of ND 15 rapidly spreads during the tensile process, causing rapid fracture of the hydrogel at a small strain (less than 20%). Combined with the calculated fracture toughness shown in Figure 6, ND 15 hydrogel shows poor fracture toughness. In short, ND 15 hydrogel without directional freezing has low mechanical strength and low toughness, so it fractures at a small strain and is not suitable for subsequent heat tensile tests. In other words, the directional freezing step is a prerequisite for heat stretching.

[0080] In addition, in the case of DF 15 prepared by directional freezing, if directional freezing is not performed according to the direction of directional freezing during tension, excellent mechanical properties cannot be obtained, it is difficult to stretch, and it is easy to break. In the case of DF 15 prepared by directional freezing, due to the directionally arranged pipe structure, the pipes are more likely to separate from each other. If heat tensile is not performed according to the direction of directional freezing during tension, DF 15 is easy to break, and the preparation process of the combination of heat tensile and directional freezing cannot be completed normally.

[0081] Example 4 Influence of Silk Fibroin Concentration on Silk Fibroin Hydrogel In this example, four groups of silk fibroin solutions were collected, with concentrations of 5%, 10%, 15%, and 20%, respectively. Each group was subjected to directional freezing according to the method provided in Example 1, followed by coaxial 60% directional tensile tension at 95°C to prepare silk fibroin hydrogel scaffolds. The tensile properties of the two prepared hydrogel scaffolds were examined and tested using a biomechanical testing machine. The tensile curves of the detected silk fibroin hydrogels at different concentrations are shown in Figure 8; the left-hand figure shows the relationship between tensile magnification and tensile stress, and the right-hand figure shows a comparison between Young's modulus and tensile stress. DF 15 H 95 S 60 This refers to a hydrogel prepared by heating and stretching a 15% silk fibroin hydrogel at 95°C to a tensile strength of 60%. As can be seen from Figure 8, as the concentration of silk fibroin increases, the scaffolding's directional effect becomes increasingly apparent, and the scaffolding's tensile properties improve. Theoretically, if the concentration of silk fibroin exceeds 20%, the tensile properties will improve further, but because there are limits to the freeze-drying effect, the tensile properties will actually decrease. Therefore, we determine that 15% is the best silk fibroin concentration.

[0082] Example 5: Effect of different temperatures on the performance of silk fibroin hydrogel scaffolds during heat tensile testing. In this example, a silk fibroin hydrogel was prepared using the method provided in Example 1. First, DSC detection was performed after step c, and the results are shown in Figure 9. In the first heating, using water molecules as a plasticizer, the glass transition temperature of the silk fibroin was approximately 77°C. In the second heating, since all water was lost from the scaffold and the plasticizing effect of the water molecules was lost, the glass transition temperature of the silk fibroin scaffold rose to 178°C. Therefore, in step d, four temperatures were set by changing the heating temperature during tensile tension (25°C, 60°C, 95°C, 130°C), and the maximum tensile stress of the prepared hydrogel scaffolds was examined. The mechanism of the scaffolds was tested using a biomechanical testing machine, and a mechanical comparison with the tensile curve of silk fibroin hydrogel is shown in Figure 10. Before the glass transition temperature (77°C), there was no significant difference in the tensile properties of the hydrogel scaffolds prepared at 25°C and 60°C. After the temperature rose to the glass transition temperature (77°C), the mechanical properties of the hydrogel scaffold prepared at 95°C were significantly higher than those of the other groups. When the temperature reached 130°C, the scaffold fractured when tensed to 60% due to the rapid loss of water molecules, and instead the mechanical properties decreased. These results indicate that heating and coaxial tensile tension at temperatures above the glass transition temperature can significantly improve mechanical properties, but this effect decreases significantly as water molecules are lost as the glass transition temperature increases. Therefore, it is determined that 95°C is a better heating temperature.

[0083] Example 6: Effect of different tensile magnifications of heated tension on the performance of silk fibroin hydrogel scaffolds In this embodiment, a silk fibroin hydrogel was prepared using the method provided in Example 1. In step d, four tensile strengths were set and the hydrogel was pulled using different tensile strengths (0%, 30%, 60%, 90%) to determine the maximum tensile stress of the prepared hydrogel scaffold. The mechanical properties of the scaffold were detected using a biomechanical testing machine. Figure 11 shows the tensile curves and mechanical comparisons of the silk fibroin hydrogel as the tensile strength increased from 0% to 60%. The mechanical properties of the scaffold gradually increased, indicating that the directional effect of silk fibroin increased as the tensile strength increased. When the tensile strength was further increased to 90%, the excessively high tensile strength caused partial fracture of the scaffold, and the mechanical properties of the scaffold decreased. Therefore, it was determined that a tensile strength of 60% was optimal.

[0084] Example 7 Directional freezing and heat tensile strength that have a synergistic effect on improving the mechanical properties of the material This example relates to three groups of silk fibroin hydrogels, which were obtained by the three preparation methods described in Example 1, Example 2, and Example 4, respectively, and they are ND 15 , DF 15 and DF 15 H 95 S 60 Includes hydrogels. Figure 12 shows the tensile stress-strain curves and the maximum tensile stress and Young's modulus obtained statistically for three groups of hydrogels. ND 15 and DF 15 Comparing the methods, and assuming that the heating and tensile step is omitted, it can be concluded that directed freezing alone can increase the maximum stress and Young's modulus of silk fibroin hydrogel by approximately 6 times and 1 time, respectively. DF 15 Further heating and tensile stress is applied to the hydrogel, as shown in Figure 12, DF 15 H 95 S 60 The maximum stress and Young's modulus are simply directional freezing DF 15 Compared to hydrogels, the maximum stress increases by approximately 8 and 7 times, and compared to the ND group, the Young's modulus increases by approximately 58 and 15 times.

[0085] Furthermore, if neither directional nor omnidirectional freezing is performed, direct heat tensile testing will make the material difficult to tensile and prone to tearing.

[0086] Therefore, directional freezing and thermal tensile treatment have a synergistic effect in improving the mechanical properties of non-directional hydrogels.

[0087] Example 8: Repair effect of silk fibroin hydrogel obtained under optimal conditions in a rabbit tendon rupture model. DF 15 H 95 S 60 Hydrogels have tensile strength and Young's modulus similar to tendons, and DF is present in the body. 15 H 95 S 60 Because the hydrogel decomposes very slowly, stable mechanical properties can be obtained over a long period of time, making it promising as a substitute for artificial tendons. DF 15 H 95 S 60 To investigate the potential of hydrogel as a tendon patch, rabbit Achilles tendons were severed, and DF was applied to the tendon stump to act as a splint, providing mechanical compensation and a growth template while maintaining tendon tension. 15 H 95 S 60 The hydrogel is sutured. As shown in Figure 13, in the initial stage of repair (4 weeks), new tendons (blue arrows) appeared in all groups, but the new tendons in the ruptured group were arranged in a disordered manner, the cell nuclei were more numerous and rounder, more angiogenesis was observed, and partial steatosis occurred (yellow arrows), DF 15 H 95 S 60 The new tendons of the group are basically DF 15 H 95 S 60 They are arranged parallel to each other in the same direction, and the cell nuclei are basically spindle-shaped, and these morphologies are more similar to normal tendons. 15 H 95 S 60 It was found that only a thin layer of fibrosis and immune reaction zones existed around it, which indicates DF 15 H 95 S 60 It exhibits good biocompatibility. DF 15 H95 S 60 The tendons themselves show little degradation and maintain high integrity (white arrows). Due to the lack of sufficient mechanical conditions, the ND group experienced rupture / displacement and could not provide effective mechanical support, and the new tendons were similar to those in the ruptured group. At week 12, the new tendons in the ND and ruptured groups were arranged in a large wave pattern, whereas in the DF group... 15 H 95 S 60 The tendons in the group were even cleaner. Up to week 48, DF 15 H 95 S 60 The new tendons in this group are very similar to normal tendons, and there are no obvious pathological differences from normal tendons.

[0088] The inventions shown and described herein can be practiced without any elements or limitations specifically disclosed herein. The terms and expressions used are intended for illustrative purposes only and not to limit, and the use of these terms and expressions is not intended to exclude similar features or parts thereof as illustrated and described, and it will be recognized that various modifications are possible within the scope of the invention. Accordingly, it should be understood that although the invention has been specifically disclosed with respect to various embodiments and selectable features, those skilled in the art can adopt modifications and variations of the concepts described herein, which are considered to fall within the scope of the invention as defined by the appended claims.

[0089] The content of all articles, patents, patent applications, and other documents and electronically available information described or recorded herein is incorporated herein by reference in whole to the same extent as individual publications are specifically and individually indicated to be incorporated by reference. The applicant reserves the right to incorporate into this application any material and information from such articles, patents, patent applications, or other documents.

Claims

1. A method for preparing a hydrogel, Step (1) involves performing directional freezing on a solution containing silk fibroin to change the solvent from a liquid state to a solid state, Step (2) involves sublimating the solvent in the product obtained in step (1) from a solid state to a gaseous state, Step (3) involves performing directional heating and tensile stress on the product obtained in step (2) to obtain the hydrogel, Step (2) further includes the step of crosslinking the silk fibroin, Step (2) further comprises a step of hydrating the crosslinked silk fibroin, wherein the solvent is the same as the solvent used to dissolve the silk fibroin in step (1), and is water. The method for preparing a hydrogel is characterized in that the directional heating and tensile in step (3) involves heating the hydrated silk fibroin hydrogel to a temperature above its glass transition temperature and 100°C or below, followed by coaxial tension, and the direction of the coaxial tension coincides with the direction of directional freezing.

2. The preparation method according to claim 1, wherein the water is pure water or mineral water.

3. The preparation method according to claim 1, comprising step (1) providing a freezing source that changes the solvent in the silk fibroin solution from a liquid state to a solid state, and bringing the silk fibroin solution close to the freezing source, thereby solidifying the silk fibroin solution starting from the direction of approaching the freezing source and moving away from the freezing source.

4. The preparation method according to claim 1, characterized in that step (1) involves injecting a silk fibroin solution into a cavity having an opening and performing directional freezing on the solution in the cavity.

5. The preparation method according to claim 4, characterized in that the temperature at which the water changes from a liquid state to ice is 0°C or lower.

6. The preparation method according to claim 5, characterized in that the temperature is less than -20°C.

7. The preparation method according to claim 6, further comprising step (1) of providing a metal rod, wherein one end of the metal rod is in contact with or close to the solution in the cavity and the other end is in contact with liquid nitrogen.

8. The preparation method according to claim 7, wherein one side of the metal rod covers the bottom of the cavity.

9. The preparation method according to claim 1, characterized in that the crosslinking is chemical crosslinking or enzymatic crosslinking.

10. The preparation method according to claim 1, wherein the sublimation method in step (2) includes a freeze-drying method, the freeze-drying method comprising the steps of: freeze-drying an ice block containing silk fibroin in a freeze-dryer, then immersing it in anhydrous ethanol to crosslink it, and then immersing it in water to completely hydrate it; or directly crosslinking an ice block containing silk fibroin, then sublimating the solvent from a solid state to a gaseous state, and then immersing it in water to completely hydrate it.

11. The preparation method according to claim 1, wherein the glass transition temperature is less than 80°C.

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