Silk nanofiber porous scaffold and preparation method thereof

Abstract

The invention provides a silk nanofiber porous scaffold and a preparation method thereof, and belongs to the field of polymer nanofiber scaffold preparation.The preparation method comprises the steps that silk nanofibers and water are evenly mixed to obtain suspension liquid, then a cross-linking agent is added into the suspension liquid, the mixture is evenly stirred to obtain a mixed solution, and the mixed solution is dried to obtain the silk nanofiber porous scaffold. Placing the mixed solution in an environment of-20 DEG C to-4 DEG C for constant-temperature freezing treatment for 14-21 days so as to improve the efficiency of the cross-linking reaction and form a topological nanostructure in the scaffold by utilizing the freezing concentration effect of ice crystals on the nanofibers, and finally unfreezing to obtain the silk nanofiber porous scaffold with high porosity. The prepared silk nanofiber porous scaffold has good water stability, and the dissolution loss rate is lower than 20% when the silk nanofiber porous scaffold is soaked in deionized water at 37 DEG C for 24 h.

Description

Technical Field

[0001] The present invention relates to the technical field of polymer nanofiber scaffold preparation, and in particular to a water-stable silk nanofiber porous scaffold and a preparation method thereof. Background Art

[0002] Hydrogen bonds play a key role in maintaining the stability and mechanical properties of natural silk's micro- and nano-mesostructures. However, the extraction process of silk nanofibers destroys the high-density hydrogen bond network within the fibers, significantly weakening the interfacial interactions between the nanofibers. This in turn significantly reduces the binding force between the nanofibers, causing the originally tightly connected fiber structure to become loose and making it difficult for the fibers to re-bond. Therefore, scaffolds prepared with pure silk nanofibers are easily affected by external factors such as temperature, humidity, and enzymes, causing the scaffold structure to disintegrate, severely limiting the application of pure silk nanofiber scaffolds in biomedicine and other fields.

[0003] Prior art, Chinese patent publication number CN111188194B provides a method for preparing a silk fibroin / gelatin fiber-reinforced chitosan composite hemostatic material. This method uses electrospun silk fibroin / gelatin composite fibers as a reinforcement and introduces chitosan to create a physical cross-linked network, resulting in a composite porous scaffold with excellent mechanical and hemostatic properties. However, chitosan is highly hydrophilic and easily swells and dissolves in physiological environments, leading to a gradual weakening of the inter-nanofibril forces, which in turn causes the scaffold structure to disintegrate, preventing it from providing sufficient support for cell growth and matrix deposition.

[0004] Chinese patent publication number CN106237381B provides a method for preparing a porous silk fibroin micro-nanofiber scaffold. Degummed silk is soaked in an acid solution to swell the silk. The silk is then subjected to repeated freeze-thaw cycles 5-50 times to induce fibril breakage within the silk fibers, resulting in a porous silk fibroin micro-nanofiber scaffold. This method can improve the scaffold's chemical stability, physical and mechanical properties, and water stability to a certain extent, but the use of acid reduces the scaffold's biocompatibility, and the repeated freeze-thaw cycles are cumbersome.

[0005] In view of this, it is necessary to design a silk nanofiber porous scaffold and a preparation method thereof to solve the above problems. Summary of the Invention

[0006] In view of the technical problems existing in the background technology, the present application provides a silk nanofiber porous scaffold and a preparation method thereof. A water-stable silk nanofiber porous scaffold is prepared by direct freezing induction in a neutral environment through a "one-step method". This method is simple, rapid, green and mild. The prepared scaffold material contains a topological nanostructure. After being immersed in deionized water at 37°C for 24 hours, the dissolution rate is less than 20%, and it has good mechanical properties and biocompatibility.

[0007] In the first aspect, an embodiment of the present application provides a method for preparing a silk nanofiber porous scaffold, comprising the following steps: uniformly mixing silk nanofibers with water to obtain a suspension, then adding a cross-linking agent to the suspension and stirring evenly to obtain a mixed solution, placing the mixed solution in an environment of -20°C to -4°C for constant temperature freezing treatment for 14-21 days, so as to utilize the freezing concentration effect of ice crystals on the nanofibers to improve the efficiency of the cross-linking reaction and form a topological nanostructure inside the scaffold, and finally thawing to obtain a silk nanofiber porous scaffold with high porosity.

[0008] Furthermore, the mass ratio of the cross-linking agent to the suspension is (0.6-1.5):1.

[0009] Furthermore, the cross-linking agent is one or more of glycerol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, epichlorohydrin, β-phenylethanol, glutaraldehyde, and carbodiimide.

[0010] Furthermore, the preparation step of the suspension further includes uniformly mixing the silk nanofibers with water, breaking them up with a food processor or a wall breaking machine, and then magnetically stirring for 30 minutes.

[0011] Furthermore, the mass fraction of the silk nanofibers in the suspension is 0.2-5wt%.

[0012] Furthermore, the diameter of the silk nanofiber is 300-800 nm; the silk nanofiber is domestic silk or wild silk.

[0013] Furthermore, the thawing temperature is 4-65°C.

[0014] In a second aspect, an embodiment of the present application provides a porous silk nanofiber scaffold, which is prepared according to the preparation method described in any one of the aforementioned technical solutions.

[0015] Furthermore, the silk nanofiber porous scaffold contains a topological nanostructure.

[0016] Furthermore, the silk nanofiber porous scaffold is used in the fields of tissue engineering scaffolds, wound dressings, drug sustained release and biofiltration.

[0017] The beneficial effects of this application are as follows: The present application provides a silk nanofiber porous scaffold and a preparation method thereof. By introducing a cross-linking agent, the silk nanofibers and the cross-linking agent are uniformly mixed and then subjected to a constant temperature freezing treatment. The freezing concentration effect of ice crystals on the nanofibers is used to improve the efficiency of the cross-linking reaction and form a topological nanostructure. The silk nanofiber porous scaffold formed after thawing has good water stability, good mechanical properties and biocompatibility.

[0018] (1) In this application, the temperature of the constant temperature freezing treatment is -20°C to -4°C. When frozen at a higher temperature, the growth rate of ice crystals is relatively low, the squeezing effect on the pores is relatively mild, and the pore deformation is relatively small, which is conducive to the stable bonding between fibers, improving the compressive strength of the scaffold and reducing the dissolution loss rate. In addition, the ice crystals formed by freezing at a higher temperature are larger in size and more evenly distributed, resulting in larger pores left after the thawing process. The large pores on the surface of the scaffold are conducive to the rapid discharge of water molecules that have invaded the interior, thereby reducing the residence time of water molecules inside the scaffold and further reducing the dissolution loss rate.

[0019] (2) In this application, the constant temperature freezing treatment time is 14-21 days. On the one hand, extending the freezing time can fully freeze the water molecules in the material and form a more stable ice crystal structure. In this way, the ice crystals melt more slowly during the thawing process, which can reduce the impact of water molecules on the fibers during rapid thawing, thereby avoiding the fiber structure from being destroyed during the thawing process and improving the stability of the scaffold. On the other hand, the more stable ice crystal structure formed by extending the freezing time will improve the stability of the scaffold by affecting the arrangement, exchange, swelling and shrinkage of the fibers, optimizing the structure and connectivity of the pores. More specifically, the freezing of water molecules will promote the interaction between the fibers, and as the freezing time increases, the fibers will be more tightly bound, forming a more stable network structure. This stable structure gives the scaffold a higher compressive strength. At the same time, the tight network structure inside the scaffold makes it difficult for water molecules to penetrate into the fibers and erode during the immersion process, thereby reducing the dissolution loss rate.

[0020] (3) In this application, the mass ratio of the cross-linking agent to the suspension is (0.6-1.5):1. Too little cross-linking agent will make the cross-linking network formed between the silk nanofibers relatively sparse, and the connection force between the fibers will not be strong enough, which will lead to a decrease in the mechanical properties of the scaffold (such as compressive strength and elastic modulus), poor stability, accelerated degradation rate, and affect the adhesion and proliferation of cells on the surface of the material. Too much cross-linking agent will make the cross-linking network formed between the fibers too dense. On the one hand, it will make it difficult to disperse the stress of the scaffold when it is suddenly impacted and it will be easily damaged. On the other hand, it will severely restrict the movement of the molecular chain segments of the scaffold, which may not only affect the effect of tissue repair, but also reduce the activity and metabolic level of cells.

[0021] The preparation method provided in this application does not require silk fibroin as a substrate. It can utilize the internal cross-linking of pure silk fibers to spontaneously form high porosity and form a topological nanostructure inside the scaffold. This is beneficial to improving the water stability of the silk nanofiber porous scaffold, providing cells with necessary adhesion sites and topographic guidance signals, and facilitating cell migration, proliferation and infiltration inside the scaffold, providing cells with a good support and growth microenvironment.

[0022] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] To more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings used in this application. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0024] Figure 1 This is a morphology diagram of the porous silk nanofiber scaffold prepared in Example 1 of the present application, wherein: Figure 1 a is the morphology of the mixed solution before freezing. Figure 1 b is the morphology of the scaffold placed in water after thawing. Figure 1 c is the morphology image taken by scanning electron microscope (SEM); Figure 2 A morphology comparison diagram of the silk nanofiber porous scaffolds prepared in Example 2, Example 4, and Example 5; Figure 3 Comparison of stress-strain curves of the silk nanoporous fiber scaffolds prepared in Examples 1-8 of the present application; Figure 4 This is a comparison chart of the CLSM imaging of the silk nanoporous fiber scaffold prepared in Example 1 after inoculation of fibroblasts and the OD values ​​obtained after culturing for 1, 3, and 7 days. DETAILED DESCRIPTION

[0025] The following embodiments of the technical solution of the present application will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application and are therefore only examples and are not intended to limit the scope of protection of the present application.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned figure descriptions are intended to cover non-exclusive inclusions.

[0027] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be understood to indicate or imply relative importance or implicitly specify the quantity, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, the meaning of "plurality" is more than two, unless otherwise clearly and specifically defined.

[0028] References herein to "embodiments" mean that a particular feature, structure, or characteristic described in connection with the embodiments may be included in at least one embodiment of the present application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor does it constitute an independent or alternative embodiment that is mutually exclusive of other embodiments. It is understood, both explicitly and implicitly, by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0029] In the description of the embodiments of this application, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this document generally indicates that the associated objects are in an "or" relationship. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of this application can be understood according to specific circumstances.

[0030] The extraction process of silk nanofibers destroys the high-density hydrogen bond network within the fibers, significantly weakening the interfacial interactions between the nanofibers. This in turn significantly reduces the binding strength between the nanofibers, loosening the previously tightly connected fiber structure and making it difficult for the fibers to re-bond. Consequently, scaffolds made from pure silk nanofibers are susceptible to external factors such as temperature, humidity, and enzymes, leading to structural disintegration, severely limiting their application in biomedical fields.

[0031] To address the generally poor water stability of silk nanofiber scaffolds, this application provides a porous silk nanofiber scaffold and a method for preparing the same. By introducing a crosslinking agent, the resulting mixed solution of silk nanofibers and the crosslinking agent is uniformly mixed and then placed in a constant-temperature freezing treatment at -20°C to -4°C for 14-21 days. The freeze-concentration effect of ice crystals on the nanofibers improves the efficiency of the crosslinking reaction and forms a topological nanostructure. The resulting porous silk nanofiber scaffold, formed after thawing, is water-stable, exhibiting a dissolution loss rate of less than 20% after immersion in deionized water at 37°C for 24 hours. The porous silk nanofiber scaffold prepared in this application also exhibits excellent mechanical properties and biocompatibility.

[0032] In the first aspect, an embodiment of the present application provides a method for preparing a silk nanofiber porous scaffold, comprising the following steps: uniformly mixing silk nanofibers with water to obtain a suspension, then adding a cross-linking agent to the suspension and stirring evenly to obtain a mixed solution, placing the mixed solution in an environment of -20°C to -4°C for constant temperature freezing treatment for 14-21 days, so as to utilize the freezing concentration effect of ice crystals on the nanofibers to improve the efficiency of the cross-linking reaction and form a topological nanostructure inside the scaffold, and finally thawing to obtain a silk nanofiber porous scaffold with high porosity.

[0033] During the preparation of the silk nanofiber porous scaffold, the temperature of the constant temperature freezing treatment should not be too low, and the treatment time should not be too short. If the freezing temperature is lower than -20°C or the freezing time is less than 14 days, the scaffold will not be formed and cannot be used.

[0034] In terms of freezing time, on the one hand, extending the freezing time can fully freeze the water molecules in the material and form a more stable ice crystal structure. In this way, the ice crystals melt more slowly during the thawing process, which can reduce the impact of water molecules on the fibers during rapid thawing, thereby avoiding the destruction of the fiber structure during the thawing process and improving the stability of the scaffold. On the other hand, the more stable ice crystal structure formed by extending the freezing time will improve the stability of the scaffold by affecting the arrangement, exchange, swelling and contraction of the fibers, optimizing the structure and connectivity of the pores. More specifically, the freezing of water molecules will enhance the interaction between the fibers, and as the freezing time increases, the fibers will be more tightly bound, forming a more stable network structure. This stable structure gives the scaffold a higher compressive strength. At the same time, the tight network structure inside the scaffold makes it difficult for water molecules to penetrate deep into the fibers and erode during the immersion process, thereby reducing the dissolution loss rate.

[0035] In terms of freezing temperatures, when frozen at higher temperatures, the growth rate of ice crystals is relatively slow, the squeezing effect on pores is relatively mild, and pore deformation is relatively small, which is conducive to stable bonding between fibers, improving the compressive strength of the scaffold and reducing the dissolution loss rate. In addition, the ice crystals formed by freezing at higher temperatures are larger and more evenly distributed, resulting in larger pores left after the thawing process. The large pores on the surface of the scaffold facilitate the rapid expulsion of water molecules that have invaded the interior, thereby reducing the residence time of water molecules inside the scaffold and further reducing the dissolution loss rate.

[0036] The preparation method provided in this application does not require silk fibroin as a substrate. It can utilize the internal cross-linking of pure silk fibers to spontaneously form high porosity and form a topological nanostructure inside the scaffold. This is beneficial to improving the water stability of the silk nanofiber porous scaffold, providing cells with necessary adhesion sites and topographic guidance signals, and facilitating cell migration, proliferation and infiltration inside the scaffold, providing cells with a good support and growth microenvironment.

[0037] In the embodiment of the present application, the preparation step of the suspension further includes uniformly mixing the silk nanofibers with water, breaking them up with a food processor or a wall breaking machine, and then magnetically stirring for 30 minutes.

[0038] In the embodiment of the present application, the mass fraction of the silk nanofibers in the suspension is 0.2-5wt%.

[0039] In the embodiment of the present application, the diameter of the silk nanofiber is 300-800 nm. The silk nanofiber is domestic silk or wild silk.

[0040] The preparation method of the silk nanofibers used in the embodiment of the present application is to add the cocoon pieces after impurities removal to a 0.5% anhydrous sodium carbonate solution and boil until the cocoon pieces are dispersed into a single fiber state, rinse thoroughly with deionized water, boil and wash again to fully remove the residual chemical reagents and sericin on the silk fibers, and then place the silk fibers at 60 ° C to dry. Next, the silk fibers are chopped and placed in a wall breaking machine for mechanical stripping for 90 minutes. Then, the fiber solution obtained after mechanical stripping is filtered three times with a 30-mesh filter, and the fibers are placed in a -80 ° C refrigerator and frozen for 7-120 hours. Finally, freeze-dried for 3-21 days to obtain silk nanofibers with a diameter of 300-800 nm.

[0041] In the embodiment of the present application, the mass ratio of the cross-linking agent to the suspension is (0.6-1.5): 1. Too little cross-linking agent will make the cross-linked network formed between the silk nanofibers relatively sparse, and the connection force between the fibers will not be strong enough, which will lead to a decrease in the mechanical properties of the scaffold (such as compressive strength and elastic modulus), poor stability, and accelerated degradation rate, while affecting the adhesion and proliferation of cells on the surface of the material. Too much cross-linking agent will make the cross-linked network formed between the fibers too dense. On the one hand, it will cause the scaffold to be difficult to disperse stress when it is suddenly impacted and easily damaged. On the other hand, the movement of the molecular segments of the scaffold will be severely restricted, which may not only affect the effect of tissue repair, but also reduce the activity and metabolic level of cells.

[0042] In the embodiment of the present application, the cross-linking agent is one or more of glycerol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, epichlorohydrin, β-phenylethanol, and glutaraldehyde.

[0043] In the embodiment of the present application, the thawing temperature is 4-65°C.

[0044] In a second aspect, an embodiment of the present application provides a porous silk nanofiber scaffold, which is prepared by any one of the preparation methods in the aforementioned technical solutions.

[0045] In the embodiment of the present application, the silk nanofiber porous scaffold contains a topological nanostructure and is immersed in deionized water at 37°C for 24 hours, and the dissolution rate is less than 20%. Preferably, the dissolution rate is less than 10%, and it has excellent water stability.

[0046] In the embodiments of the present application, the silk nanofiber porous scaffold is used in the fields of tissue engineering scaffolds, wound dressings, drug sustained release and biofiltration.

[0047] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used without manufacturer's indication are all commercially available conventional products.

[0048] Example 1 Example 1 provides a method for preparing a porous silk nanofiber scaffold, comprising the following steps: Tussah silk nanofiber blocks with a fiber diameter of 300-800nm ​​were added to deionized water and stirred evenly with a wall breaker to prepare a suspension with a concentration of 0.8wt%. Next, 1,4-butanediol diglycidyl ether was added to the suspension and stirred evenly to obtain a mixed solution. The mixed solution was placed in a -4°C environment and frozen for 21 days. The freeze-concentration effect of ice crystals on the nanofibers was used to improve the efficiency of the cross-linking reaction to form a topological nanostructure. The solution was then thawed at room temperature (25°C) to obtain a high-porosity silk nanofiber porous scaffold. The mass ratio of the cross-linking agent 1,4-butanediol diglycidyl ether to the suspension was 1.5:1.

[0049] See also Figure 1 As shown, Figure 1 a is the morphology of the mixed solution before freezing. Figure 1 b shows the morphology of the silk nanofiber porous scaffold placed in water after thawing. It can be seen that the structure of the scaffold remains stable in water. Figure 1 c is the morphology of the silk nanofiber porous scaffold taken by scanning electron microscope (SEM). It can be seen that the scaffold has a complex network structure formed by the fibers interweaving with each other, with high porosity and showing the characteristics of a topological structure. This complex network structure enables a large number of contact points and interaction forces between the fibers, which can improve the stability of the scaffold to a certain extent.

[0050] Example 2 The difference between Example 2 and Example 1 is that the mixed solution is placed in a -4°C environment for constant temperature freezing treatment for 14 days. Other details are the same as in Example 1 and will not be repeated here.

[0051] Example 3 The difference between Example 3 and Example 1 is that the mixed solution is placed in a -4°C environment for constant temperature freezing treatment for 18 days. Other details are the same as in Example 1 and will not be repeated here.

[0052] Example 4 The difference between Example 4 and Example 1 is that the mixed solution is placed in a -12°C environment for constant temperature freezing treatment for 14 days. Other details are the same as in Example 1 and will not be repeated here.

[0053] Example 5 The difference between Example 5 and Example 1 is that the mixed solution is placed in a -20°C environment for constant temperature freezing treatment for 14 days. The rest is the same as Example 1 and will not be repeated here.

[0054] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the mixed solution is placed in a -4°C environment for constant temperature freezing treatment for 7 days. Other details are the same as in Example 1 and will not be repeated here.

[0055] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the mixed solution is placed in a -20°C environment for constant temperature freezing treatment for 7 days. Other details are the same as in Example 1 and will not be repeated here.

[0056] Example 6 The difference between Example 6 and Example 1 is that the mass ratio of the cross-linking agent 1,4-butanediol diglycidyl ether to the suspension is 0.6:1. Other details are the same as in Example 1 and will not be repeated here.

[0057] Example 7 The difference between Example 7 and Example 1 is that the mass ratio of the cross-linking agent 1,4-butanediol diglycidyl ether to the suspension is 0.9:1. The other details are the same as those in Example 1 and will not be repeated here.

[0058] Example 8 The difference between Example 8 and Example 1 is that the tussah silk nanofiber block is replaced with the Bombyx mori silk nanofiber block. The rest is the same as Example 1 and will not be repeated here.

[0059] The morphology comparison of the silk nanofiber porous scaffolds prepared in Example 2, Example 4 and Example 5 is shown in FIG. Figure 2 As shown in the figure, it can be seen that as the freezing temperature increases (from -4°C to -20°C), the pores of the scaffold become significantly smaller. This is because the porosity of the scaffold is affected by the size, growth direction, and arrangement density of the ice crystals. Some ice crystals fill the pores and squeeze the pore walls during growth, causing the pore structure to change. At relatively high freezing temperatures, the growth rate of ice crystals is relatively slow, the size of the ice crystals formed is relatively large, and the pores left after thawing are also larger. At the same time, when ice crystals grow at higher temperatures, the squeezing effect on the pores is relatively mild, and the pore deformation is relatively small, which is conducive to the retention of large-sized pores. Relatively low freezing temperatures will result in smaller and irregular pores.

[0060] The silk nanoporous fiber scaffolds prepared in Examples 1-8 were placed in a container filled with deionized water, and the container was placed in a constant temperature water bath at 37°C and shaken for 24 hours, and then dried. The dry weight change of the silk nanoporous fiber scaffolds before and after soaking was tested, and the dissolution loss rate was calculated.

[0061] The silk nanoporous fiber scaffolds prepared in Examples 1-8 were subjected to uniaxial compression tests using a universal material testing machine to obtain stress-strain curves, compression strength, and compression modulus. For a comparison of stress-strain curves, see Figure 3 As shown, the curves of Example 1 and Example 8 overlap.

[0062] The scaffolds prepared in Comparative Example 1-2 were not formed and could not be used for dissolution loss rate and mechanical property tests.

[0063] For the convenience of comparison, the test data of the dissolution rate, compressive strength and compression modulus of the silk nanoporous fiber scaffolds are summarized in the table below.

[0064] By comparison, it can be seen that prolonging the constant temperature freezing treatment time, increasing the constant temperature freezing treatment temperature or increasing the cross-linking agent content will reduce the dissolution rate of the silk nanoporous fiber scaffold and increase the compressive strength and compression modulus of the scaffold.

[0065] In terms of freezing time, on the one hand, extending the freezing time can fully freeze the water molecules in the material and form a more stable ice crystal structure. In this way, the ice crystals melt more slowly during the thawing process, which can reduce the impact of water molecules on the fibers during rapid thawing, thereby avoiding the destruction of the fiber structure during the thawing process and improving the stability of the scaffold. On the other hand, the more stable ice crystal structure formed by extending the freezing time will improve the stability of the scaffold by affecting the arrangement, exchange, swelling and contraction of the fibers, optimizing the structure and connectivity of the pores. More specifically, the freezing of water molecules will enhance the interaction between the fibers, and as the freezing time increases, the fibers will be more tightly bound, forming a more stable network structure. This stable structure gives the scaffold a higher compressive strength. At the same time, the tight network structure inside the scaffold makes it difficult for water molecules to penetrate deep into the fibers and erode during the immersion process, resulting in a lower dissolution rate.

[0066] In terms of freezing temperatures, when frozen at higher temperatures, the growth rate of ice crystals is relatively slow, the squeezing effect on pores is relatively mild, and pore deformation is relatively small, which is conducive to stable bonding between fibers, improving the compressive strength of the scaffold and reducing the dissolution loss rate. In addition, the ice crystals formed by freezing at higher temperatures are larger and more evenly distributed, resulting in larger pores left after the thawing process. The large pores on the scaffold surface facilitate the rapid expulsion of water molecules that have invaded the interior, thereby reducing the residence time of water molecules inside the scaffold and reducing the dissolution loss rate.

[0067] In terms of the amount of cross-linking agent used, appropriately increasing the amount of cross-linking agent can significantly enhance the degree of cross-linking between silk nanofibers, forming a tighter cross-linking network, increasing the stability of the scaffold, thereby reducing the dissolution rate and improving the compressive strength.

[0068] The in vitro biocompatibility test of the silk nanoporous fiber scaffold prepared in Example 1 was performed using fibroblasts, comprising the following steps: S1, material sterilization. That is, the silk nanofiber membrane was prepared into a thin sheet with a diameter of 15mm and a thickness of 1-2mm, soaked in alcohol for 15-30 minutes, and then placed in a clean bench for ultraviolet sterilization for 1 hour; S2, cell recovery. That is, the frozen seed cells were removed from the liquid nitrogen tank and thawed in a 37°C water bath. The supernatant was then removed and complete medium was added. After pipetting and vortexing, the cells were poured into a cell culture flask and complete medium was added again. S3, cell passage. First, wash the cell culture flask twice with PBS buffer. Add trypsin to dissociate the cells from the culture vessel surface and free them. Next, discard the trypsin, add complete medium, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant, then add complete medium and swirl evenly. Pour the supernatant into a new culture flask, and add complete medium again.

[0069] S4, cell seeding. That is, after the cell density reaches 80-90% confluence, the cells are digested with trypsin, centrifuged and evenly pipetted and counted, and then the cells are diluted to 5×10 with complete culture medium. 4 Then, cells were seeded on the surface of the silk nanoporous fiber scaffold at 100 μL / well, and 1 mL of complete culture medium was added and CO 2 Cultured in an incubator, the medium was changed at intervals during the culture process, and images were taken using a confocal laser scanning microscope (CLSM) on the 1st, 3rd, and 7th day of culture, as well as CCK-8 tests. The imaging and test results are shown in Figure 4 shown.

[0070] From the CLSM imaging, it can be seen that the optical density gradually increases with the extension of culture time, and the morphology of fibroblasts at each time point is clearly visible. The OD value obtained by the CCK-8 test also reflects that the number of cells increases significantly with the extension of culture time. This shows that the silk nanoporous fiber scaffold prepared in this application has good biocompatibility and can support the normal growth of fibroblasts.

[0071] In summary, the present application provides a silk nanofiber porous scaffold and a preparation method thereof, which utilizes the freezing concentration effect of ice crystals on nanofibers to improve the efficiency of the cross-linking reaction and form a topological nanostructure inside the scaffold. At the same time, the freezing temperature, freezing time and amount of cross-linking agent are controlled, so that the final silk nanofiber porous scaffold has good stability, low dissolution rate and high compressive strength.

[0072] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A method for preparing a porous silk nanofiber scaffold, characterized in that: The method comprises the following steps: uniformly mixing silk nanofibers with water to obtain a suspension, then adding a crosslinking agent to the suspension and stirring evenly to obtain a mixed solution, placing the mixed solution in an environment of -20°C to -4°C for constant temperature freezing treatment for 14-21 days, so as to utilize the freezing concentration effect of ice crystals on the nanofibers to improve the efficiency of the crosslinking reaction and form a topological nanostructure inside the scaffold, and finally thawing to obtain a high-porosity silk nanofiber porous scaffold.

2. The method for preparing a porous silk nanofiber scaffold according to claim 1, wherein: The mass ratio of the cross-linking agent to the suspension is (0.6-1.5):

1.

3. The method for preparing a porous silk nanofiber scaffold according to claim 2, wherein: The cross-linking agent is one or more of glycerol diglycidyl ether, polypropylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, epichlorohydrin, β-phenylethanol, glutaraldehyde, and carbodiimide.

4. The method for preparing a porous silk nanofiber scaffold according to claim 1, wherein: The preparation step of the suspension further includes uniformly mixing the silk nanofibers with water, breaking them up with a food processor or a wall breaking machine, and then magnetically stirring for 30 minutes.

5. The method for preparing a porous silk nanofiber scaffold according to claim 4, wherein: The mass fraction of the silk nanofibers in the suspension is 0.2-5 wt %.

6. The method for preparing a porous silk nanofiber scaffold according to claim 1, wherein: The diameter of the silk nanofiber is 300-800nm; the silk nanofiber is domestic silk or wild silk.

7. The method for preparing a porous silk nanofiber scaffold according to claim 1, wherein: The thawing temperature is 4-65℃.

8. A porous silk nanofiber scaffold, characterized in that: The silk nanofiber porous scaffold is prepared according to the preparation method described in any one of claims 1-7.

9. The porous silk nanofiber scaffold according to claim 8, characterized in that: The silk nanofiber porous scaffold contains a topological nanostructure.

10. The porous silk nanofiber scaffold according to claim 9, characterized in that: The silk nanofiber porous scaffold is used in the fields of tissue engineering scaffolds, wound dressings, drug sustained release and biological filtration.

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