High-elasticity chitosan / silk fibroin semi-interpenetrating network hydrogel and photo-curing preparation method thereof

Abstract

The application relates to a high-elasticity chitosan / silk fibroin semi-interpenetrating network hydrogel and a photo-curing preparation method thereof. The high-elasticity chitosan / silk fibroin semi-interpenetrating network hydrogel is obtained by mixing a regenerated silk fibroin solution and a solution containing acylated chitosan, then performing ultrasonic treatment and photo-curing, the compressive stress is 1000-1200 kPa, the maximum strain is 50-60%, and the Young's modulus is 300-400 MPa. The high-elasticity chitosan / silk fibroin semi-interpenetrating network hydrogel provided by the application has good mechanical properties, elasticity and biocompatibility, and has a good application prospect in the field of biomedical materials.

Description

Technical Field

[0001] This invention belongs to the field of polymer gel technology, specifically relating to a highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel and its photocuring preparation method. Background Technology

[0002] Hydrogels are a class of highly hydrophilic three-dimensional network gels that swell rapidly in water without dissolving. They are typically formed from hydrophilic polymers through physical or chemical cross-linking. Hydrogels possess excellent biocompatibility and degradability, low immunogenicity, and easily modifiable mechanical properties. Therefore, hydrogels can serve not only as tissue scaffolds but also as sustained-release carriers in neural tissue engineering, promoting cell proliferation, migration, and differentiation, and accelerating the repair of injured tissues. However, the relatively poor mechanical properties of existing hydrogels limit their application scope.

[0003] Chitosan possesses excellent biocompatibility, biodegradability, hemostatic properties, mucosal adsorption, non-toxicity, and antibacterial properties, making it one of the most common materials for preparing biomedical hydrogels. Silk fibroin also exhibits good biocompatibility and mechanical properties, thus finding application in the preparation of many biomedical materials. Pure silk fibroin hydrogels and pure chitosan hydrogels generally have poor mechanical properties, which can be improved through physical and chemical crosslinking. While chemical crosslinking offers better mechanical properties than physical crosslinking, it can introduce toxic substances, affecting the material's cellular compatibility and limiting the application range of chitosan hydrogels.

[0004] In view of this, the present invention provides a chitosan / silk fibroin semi-interpenetrating network hydrogel, which effectively enhances the elasticity of the material, improves the mechanical properties of single-material gels, and at the same time preserves the cell compatibility of the material. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the above-mentioned deficiencies in the prior art by providing a highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel and its photocuring preparation method. The hydrogel has a stable three-dimensional network structure and exhibits good mechanical properties, elasticity and biocompatibility. It has good application prospects in the field of biomedical materials, and the preparation method is fast and simple, which can realize mass production.

[0006] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0007] A highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel is provided, which is obtained by mixing a regenerated silk fibroin (SF) solution with an acylated chitosan (CSMA) solution, followed by ultrasonic treatment and photocuring. Its compressive stress is 1000-1200 kPa, maximum strain is 50-60%, and Young's modulus is 300-400 MPa.

[0008] According to the above scheme, the concentration of the regenerated silk fibroin solution is 2.5–25 wt%. Different concentrations of the regenerated silk fibroin solution are selected based on the intended use of the product. When the concentration of the regenerated silk fibroin solution is 2.5–6 wt%, the resulting hydrogel exhibits good mechanical properties and biocompatibility, and has potential application value in biomedical and tissue surgical implantation fields. When the concentration of the regenerated silk fibroin solution is 15–25 wt%, the resulting hydrogel, after freezing, alcohol soaking, and drying, can be used as an artificial bone screw.

[0009] According to the above scheme, the concentration of acylated chitosan in the acylated chitosan solution is 1-5 wt%.

[0010] According to the above scheme, the solution containing acylated chitosan also contains a photoinitiator. A preferred photoinitiator is Irgacure 2959 (I2959).

[0011] According to the above scheme, the mass ratio of silk fibroin in the silk fibroin solution to acylated chitosan in the acylated chitosan solution is 1 to 10:1.

[0012] According to the above scheme, the process conditions for ultrasonic treatment are: ultrasonic power 100-200W, ultrasonic treatment time 3-60s.

[0013] According to the above scheme, the photocuring conditions are as follows: the photocuring reaction is carried out under ultraviolet lamp irradiation, the ultraviolet lamp power is 100-900W, and the photocuring reaction time is 5-10min.

[0014] This invention also provides a method for preparing the above-mentioned highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel, the specific steps of which are as follows:

[0015] 1) Preparation of regenerated silk fibroin solution: Disperse the silk fibroin fibers obtained by degumming and drying silk in LiBr solution, heat and stir thoroughly, then dialyze (to remove salt ions), filter and centrifuge the dialyzed silk fibroin solution, and dilute or concentrate as needed to obtain regenerated silk fibroin solution;

[0016] 2) Preparation of acylated chitosan: Chitosan was added to deionized water and mixed well. The mixture was heated and stirred thoroughly. Then, acetic acid solution was added and stirred evenly. Finally, methacrylic anhydride was added and stirred for 3-6 hours. The reaction solution was taken out and cooled before dialyzing (to remove unreacted methacrylic anhydride). The dialyzed solution was filtered, centrifuged, and pre-cooled. Then, it was freeze-dried to obtain acylated chitosan.

[0017] 3) Preparation of highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel: First, prepare a photoinitiator Irgacure 2959 solution, then disperse the acylated chitosan obtained in step 2) in the photoinitiator Irgacure 2959 solution to obtain a solution containing acylated chitosan, then mix it with the regenerated silk fibroin solution obtained in step 1) in proportion and stir evenly in the dark, then sonicate the resulting blend solution, and then place it under a UV lamp for light curing to obtain a highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel.

[0018] According to the above scheme, the silk degumming method in step 1) is as follows: add silk to deionized water, heat to boiling, add Na2CO3, continue boiling for 30-60 minutes, then thoroughly wash the silk with deionized water, and repeat this process 2-3 times.

[0019] According to the above scheme, the mass ratio of silk to deionized water is 1:25 to 100, and the concentration of Na2CO3 in the deionized water is 0.05 to 0.1 wt%.

[0020] According to the above scheme, the concentration of the LiBr solution in step 1) is 9.3M (mol / L), and the concentration of silk fibroin in the LiBr solution is 0.1-0.3 g / mL.

[0021] According to the above scheme, the heating temperature in step 1) is 50-60℃.

[0022] According to the above scheme, the concentration method in step 1) is as follows: the dilute regenerated silk fibroin solution (concentration of 2.5-6 wt%) obtained after centrifugation is transferred to a dialysis bag and immersed in a polyethylene glycol solution (number average molecular weight of 10,000-20,000) with a concentration of 20-30 wt%. When the immersion time is 24-48 hours, the concentration of the obtained regenerated silk fibroin solution is 15-25 wt%.

[0023] According to the above scheme, the chitosan in step 2) has a number-average molecular weight of 10-20W, a degree of deacetylation of 80-95%, and a concentration of chitosan in deionized water of 1-4wt%. The acylation rate of the obtained acylated chitosan is approximately 20-50%.

[0024] According to the above scheme, the heating temperature in step 2) is 50-60℃.

[0025] According to the above scheme, the concentration of the acetic acid solution in step 2) is 1-3% (v / v), and the mass-to-volume ratio of chitosan to acetic acid solution is 1g / 0.24-1.5mL.

[0026] According to the above scheme, the mass ratio of methacrylic anhydride to chitosan in step 2) is 1.4 to 8:1.

[0027] According to the above scheme, the pre-cooling conditions for step 2) are: freezing at -60 to -80°C for 8 to 12 hours. Water molecules are frozen through pre-cooling, and then sublimated in a freeze dryer to obtain sponge-like acylated chitosan.

[0028] According to the above scheme, the concentration of the photoinitiator Irgacure 2959 solution in step 3) is 0.1-0.5 wt%.

[0029] According to the above scheme, the concentration of acylated chitosan in the solution containing acylated chitosan in step 3) is 1-5 wt%.

[0030] The present invention also includes the application of the above-mentioned highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel as a tissue surgical implant material and bone repair material in the field of biomedical materials.

[0031] This invention also includes an artificial bone nail further obtained from the above-mentioned highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel. The hydrogel is obtained by mixing a 15-25 wt% regenerated silk fibroin solution with an acylated chitosan solution, followed by ultrasonic treatment and photocuring. The resulting hydrogel is then frozen (at -80°C), immersed in anhydrous alcohol for 48-72 hours, and finally dried. Freezing fixes the shape, and subsequent immersion in alcohol causes water loss within the hydrogel, completely transforming the silk fibroin into a β-sheet structure, resulting in greater stability and further improving the material's mechanical properties.

[0032] This invention employs silk fibroin and chitosan to form a semi-interpenetrating hydrogel because silk fibroin molecules are negatively charged overall, and their crystalline regions are rich in basic amino acids, exhibiting a certain adsorption effect on cells, thus giving the SF-based hydrogel good biocompatibility. Chitosan, second only to cellulose in terms of molecular weight, is the only naturally occurring positively charged basic polysaccharide. Chitosan possesses excellent biocompatibility, cell adhesion, biodegradability, and antibacterial properties. Utilizing the opposite charge between chitosan and silk fibroin, electrostatic interactions can generate polyelectrolyte complexes, further enhancing the mechanical properties of the semi-interpenetrating hydrogel. Acylated chitosan (CSMA) is obtained by modifying chitosan (CS) with methacrylic anhydride (MA). The modified CS not only improves water solubility but also endows the chitosan molecular chain with photosensitive propylene groups. The semi-interpenetrating hydrogel is obtained by mixing CSMA solution with regenerated silk fibroin (SF) solution and then ultrasonically irradiating the mixture.

[0033] This invention combines ultrasonic treatment with photocuring to prepare a CSMA / SF semi-interpenetrating network hydrogel. After mixing the regenerated silk fibroin solution with the acylated chitosan solution, the ultrasonic treatment stage induces the formation of antiparallel β-sheet structures in the silk fibroin, which promotes the entanglement of the regenerated silk fibroin and the acylated chitosan. Then, the photocuring method is used to promote the photocrosslinking polymerization reaction of the acylated chitosan, thereby forming a semi-interpenetrating hydrogel with a "brick and mortar" structure. Without adding chemical crosslinking agents, the prepared hydrogel not only has good mechanical properties, but also good biocompatibility and cell adhesion.

[0034] The beneficial effects of this invention are as follows: 1. The highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel provided by this invention has good mechanical properties, elasticity, and biocompatibility, and has good application prospects in the field of biomedical materials. 2. This invention uses silk and chitosan as raw materials, both of which are biocompatible and biodegradable polymers, making them green and environmentally friendly. Furthermore, the preparation process allows for rapid gelation, is simple to operate, and has a streamlined process, making it easy to achieve industrial production. Attached Figure Description

[0035] Figure 1 The CSMA prepared in Example 1 of this invention 50 / SF 50 Comparison of semi-interpenetrating network hydrogel before and after UV curing;

[0036] Figure 2 SEM images of the samples of hydrogels prepared in Example 1 and Comparative Examples 1-2 after freeze-drying;

[0037] Figure 3 XRD patterns of the hydrogels prepared in Example 1 and Comparative Examples 1-2;

[0038] Figure 4 FTIR images of the hydrogels prepared in Example 1 and Comparative Examples 1-2;

[0039] Figure 5 The TG, DTG, and DSC images are of the hydrogels prepared in Example 1 and Comparative Examples 1-2.

[0040] Figure 6 Compression curves and Young's modulus plots of the hydrogels prepared in Example 1 and Comparative Examples 1-2;

[0041] Figure 7 CSMA prepared in Example 1 50 / SF 50 Cyclic compression curve of semi-interpenetrating network hydrogel;

[0042] Figure 8 The images show the biocompatibility test results of the hydrogels prepared in Example 1 and Comparative Examples 1-2.

[0043] Figure 9 This is a cell compatibility test diagram of the artificial bone nail prepared in Example 2. Detailed Implementation

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0045] Comparative Example 1

[0046] A pure silk fibroin hydrogel is prepared by the following method:

[0047] S1. Preparation of regenerated silk fibroin solution:

[0048] (1) Take 100g of silkworm silk, add 5000mL of deionized water, heat to boiling, add Na2CO3, continue to boil for 30min, then thoroughly wash the silkworm silk with deionized water, repeat this degumming process 3 times, the concentration of Na2CO3 added in the deionized water during the 3 degumming processes is 0.1wt%, 0.1wt%, and 0.05wt%, respectively, place the degummed silkworm silk in a 60℃ oven and dry for 48 hours, constantly loosening it during the process, and obtain silk fibroin fiber after drying;

[0049] (2) Take 30g of silk fibroin and add it to 150mL of LiBr solution (9.3M). Place it in a 60℃ water bath to dissolve for 30min, then stir for 45min. Take out the solution, cool it, and transfer it to a dialysis bag. Dialyze it in a 4℃ refrigerator for 72h. Filter and centrifuge the dialyzed silk fibroin solution. Take the supernatant and dilute it to obtain a 5wt% regenerated silk fibroin solution. Place it in a 4℃ refrigerator for later use.

[0050] S2. Preparation of pure silk fibroin hydrogel: Take 10 mL of the regenerated silk fibroin solution obtained in S1 and place it in a beaker for ultrasonic treatment. The ultrasonic power is 100 W and the ultrasonic treatment time is 60 s. Then transfer it to a mold to form pure silk fibroin hydrogel.

[0051] Comparative Example 2

[0052] A pure acylated chitosan hydrogel is prepared by the following method:

[0053] Preparation of S1 and CSMA: 2g of chitosan (number average molecular weight 15W, degree of deacetylation 80-95%) was mixed with 98g of deionized water, placed in a 50℃ water bath and stirred for 60min, then 1.2mL of acetic acid solution (concentration 1.2% v / v) was added and stirred for 8h, and finally 7g of methacrylic anhydride was added and stirred for 4h. The reaction solution was taken out, cooled and placed in a 4℃ refrigerator for dialyzing for 72h. The dialyzed solution was filtered, centrifuged and poured into a glass dish and pre-cooled at -80℃ for 8h, and then freeze-dried in a freeze dryer for 60h to obtain acylated chitosan.

[0054] S2. Preparation of pure acylated chitosan hydrogel: First, prepare a 0.135 wt% photoinitiator Irgacure2959 solution. Take 0.5 g of the acylated chitosan obtained in S1 and disperse it in 10 mL of the photoinitiator Irgacure2959 solution. Then, place it under a UV lamp for photocrosslinking (UV lamp power 600 W, photocrosslinking time 5 min) to obtain pure acylated chitosan hydrogel.

[0055] Example 1

[0056] A highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel is prepared as follows:

[0057] S1. Preparation of regenerated silk fibroin solution:

[0058] (1) Take 100g of silkworm silk, add 5000mL of deionized water, heat to boiling, add Na2CO3, continue to boil for 30min, then thoroughly wash the silkworm silk with deionized water, repeat this degumming process 3 times, the concentration of Na2CO3 added in the deionized water during the 3 degumming processes is 0.1wt%, 0.1wt%, and 0.05wt%, respectively, place the degummed silkworm silk in a 60℃ oven and dry for 24 hours, constantly loosening it during the process, and obtain the desired silk fibroin fiber after drying.

[0059] (2) Take 30g of silk fibroin and add it to 150mL of LiBr solution (9.3M). Place it in a 60℃ water bath and dissolve it for 30min. Then stir for 45min. Take out the solution, cool it, and transfer it to a dialysis bag. Dialyze it at 4℃ for 72h. Filter and centrifuge the dialyzed silk fibroin solution. Take the supernatant and dilute it to obtain a 5wt% regenerated silk fibroin solution. Place it in a 4℃ refrigerator for later use.

[0060] S2. Preparation of acylated chitosan: 2g of chitosan (number average molecular weight 15W, degree of deacetylation 80-95%) was mixed with 98g of deionized water, placed in a 50℃ water bath and stirred for 60min, then 1.2mL of acetic acid solution (concentration 1.2% v / v) was added and stirred for 8h, and finally 7g of methacrylic anhydride was added and stirred for 4h. The reaction solution was taken out, cooled and placed in a 4℃ refrigerator for dialyzing for 72h. The dialyzed solution was filtered, centrifuged and poured into a glass dish and pre-cooled at -80℃ for 8h, and then freeze-dried in a freeze dryer for 60h to obtain acylated chitosan.

[0061] Preparation of S3, Highly Elastic Chitosan / Silk Fiber Semi-Interpenetrating Network Hydrogel: First, prepare a 0.135 wt% photoinitiator Irgacure 2959 solution. Disperse 0.25 g of the acylated chitosan obtained in S1 in 5 mL of the photoinitiator Irgacure 2959 solution, then add 5 mL of the regenerated silk fibroin solution prepared in S1. Stir evenly in the dark. The resulting blend solution is then sonicated at 100 W for 60 s. Immediately after treatment, it is placed under a 600 W UV lamp for 7 min to cure, thus obtaining the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel (CSMA). 50 / SF 50 (Semi-interpenetrating network hydrogel).

[0062] The CSMA prepared in this embodiment 50 / SF 50 The semi-interpenetrating network hydrogel was compared before and after UV curing using a 45° tilt method, and the results are as follows: Figure 1 As shown, Figure (a) is the mixed solution before curing, and Figure (b) is the actual gel. Using the 45° tilt method for observation, it can be seen that when the bottle is tilted after curing, the liquid level does not change, indicating that a gel has been formed.

[0063] SEM characterization was performed on the samples of the hydrogels prepared in this embodiment and Comparative Examples 1-2 after freeze-drying. The results are as follows: Figure 2 As shown. Figures (a) and (b) are SEM images of the pure silk fibroin hydrogel prepared in Comparative Example 1; Figures (c) and (d) are SEM images of the pure acylated chitosan hydrogel prepared in Comparative Example 2; Figures (e) and (f) are SEM images of the CSMA prepared in this example.50 / SF 50 SEM image of the semi-interpenetrating network hydrogel. Figure 2 It can be seen that the pure silk fibroin hydrogel is relatively dense, while the pure acylated chitosan hydrogel has a loose and porous sheet-like structure. In this embodiment, when the CSMA content is 50 wt%, the degree of crosslinking with SF is relatively large, resulting in smaller pores and a denser structure in the hydrogel. This is because during the chemical crosslinking process to form a semi-interpenetrating network hydrogel, the increase in CSMA content leads to an increase in the degree of crosslinking of molecular chain segments between CSMA and SF. Therefore, the CSMA content at this point is relatively high. 50 / SF 50 Semi-interpenetrating network hydrogels have smaller pore sizes.

[0064] XRD tests were performed on the hydrogels prepared in this embodiment and Comparative Examples 1-2, and the results are as follows: Figure 3 As shown in the XRD images, the pure silk fibroin (SF) hydrogel exhibits characteristic peaks at 20.7° and 24.5°, respectively. The crystallization peak at 24.5° represents the α-helix (Silk-I) structure of silk fibroin, while the crystallization peak at 20.7° represents the β-sheet (Silk-II) structure. This indicates that the silk fibroin in Comparative Example 1, after sonication, has a more stable structure compared to the untreated silk fibroin (mainly existing in the Silk-I structure), while CSMA did not show any obvious characteristic peaks. In Example 1, after the addition of CSMA, CSMA... 50 / SF 50 The semi-interpenetrating network hydrogel exhibits a distinct characteristic peak at 9.1° (representing the Silk-II structure), and the crystallization peak at 24.5° disappears, indicating that the introduction of CSMA causes the α-helical structure of SF to transform into a β-sheet structure.

[0065] The hydrogels prepared in this embodiment and Comparative Examples 1-2 were characterized by FTIR, and the results are as follows: Figure 4 As shown in Figure (a). Figure (a) shows the hydrogels prepared in this embodiment and Comparative Examples 1-2 in a scanning range of 4000–400 cm⁻¹. -1 The infrared spectrum; Figure (b) shows the infrared spectrum of the hydrogels prepared in this embodiment and Comparative Examples 1-2 in the scanning range of 2000–500 cm⁻¹. -1 The infrared spectrum of CSMA. As can be seen from (a), CSMA is in the range of 1600–1650 cm⁻¹. -1 The absorption peak of the -NH2 stretching vibration disappears at 1658 cm⁻¹, while it disappears at 1658 cm⁻¹. -1 1535cm -1 1378cm -1The characteristic absorption peaks of amide I, II, and III bands appear at [value missing], respectively. The disappearance of the amino absorption peak and the formation of the amide bond absorption peak indicate that the amino groups on MA and CS have undergone acylation. As shown in Figure (b), the characteristic infrared absorption peak of the SF hydrogel appears at 1624 cm⁻¹. -1 (α-helical structure), 1521cm -1 At the (random curl) point, CSMA 50 / SF 50 The characteristic infrared absorption peak of the semi-interpenetrating network hydrogel appears at 1657 cm⁻¹. -1 1535cm -1 The position represents the β-sheet structure of SF, indicating that the introduction of CSMA causes both the α-helix structure and random coil of SF to transform into a β-sheet structure.

[0066] The Tg of the hydrogels prepared in this embodiment and Comparative Examples 1-2 was characterized, and the results are as follows: Figure 5 As shown in Figure (a), the thermogravimetric analysis (TG) curves of the hydrogels prepared in this embodiment and Comparative Examples 1-2 at 30–1000℃ show their thermodynamic properties; Figure (b) shows the differential thermogravimetric analysis (DTG) curves of the hydrogels prepared in this embodiment and Comparative Examples 1-2 at 30–1000℃; and Figure (c) shows the DSC curves of the hydrogels prepared in this embodiment and Comparative Examples 1-2 at 30–320℃. Figure (a) shows that the first stage of weight loss occurred at 30–100℃, mainly due to the evaporation of moisture from the material, leading to a sharp decrease in mass. A second significant mass loss occurred at 250±10℃–450℃, due to the decomposition of the internal organic macromolecular structure. Figure (b) shows the CSMA, SF, and CSMA... 50 / SF 50 The maximum weight loss temperatures of the hydrogels were 266.6℃, 303.4℃, and 304.1℃, respectively. From the first stage of weight loss, combined with XRD and FTIR, it can be seen that CS, after modification with MA, has better water solubility. This is because the crystallinity of the modified CS is reduced, resulting in a higher water content and greater mass loss compared to the more crystalline SF. The mass loss in the second stage shows that the addition of CSMA leads to a more stable structure in the hydrogel, reducing the decomposition rate of organic macromolecules and thus decreasing the mass loss rate. Figure (c) shows that the glass transition temperatures of CSMA and SF hydrogels are 217.7℃ and 278.5℃, respectively; CSMA... 50 / SF 50 The hydrogel exhibits two glass transition temperatures, 221.7℃ and 296.6℃ respectively, indicating an interaction between CSMA and SF in the semi-interpenetrating network hydrogel formed after blending. 50 / SF 50Hydrogels have a more stable structure. Furthermore, DSC analysis shows that CSMA and SF are incompatible and do not interfere with each other to form a gel, thus confirming that the hydrogel provided in this embodiment is a "brick-and-mortar" structured semi-interpenetrating network hydrogel.

[0067] Compression tests were performed on the hydrogels prepared in this embodiment and Comparative Examples 1-2, and the results are as follows: Figure 6 As shown. Figure 6 (a) shows the compression curves of the hydrogels prepared in this example and Comparative Examples 1-2. The curves show the compression properties of CSMA, SF, and CSMA. 50 / SF 50 The maximum stresses were 708.4 kPa, 109.9 kPa, and 1187.8 kPa, respectively, and the maximum strains were 40.9%, 41.5%, and 51.4%, respectively. Figure 6 (b) is the Young's modulus diagram of the hydrogels prepared in this embodiment and Comparative Examples 1-2, CSMA. 50 / SF 50 The Young's modulus was 383.1 MPa, indicating that the blending of CSMA and SF not only enhanced the mechanical properties of the hydrogel but also significantly improved its resilience. It can be seen that the Young's modulus of the formed semi-interpenetrating network hydrogel was significantly enhanced, indicating the presence of intermolecular interactions between CSMA and SF molecules, which endowed the hydrogel with a more stable structure, thus resulting in excellent resilience.

[0068] The CSMA prepared in this embodiment 50 / SF 50 The semi-interpenetrating network hydrogel underwent resilience testing, and the cyclic compression-rebound curve of the hydrogel is shown below. Figure 7 As shown. The test involved three compressions: the first compression deformation was 20%, the second was 35%, and the third was 50%. Figure 7 It can be seen that the loading and unloading curves of the stress-strain curves almost overlap during the first and second compression processes, while the third loading and unloading curve is a closed curve with a small area, indicating that the material is less damaged during cyclic compression. This demonstrates that the CSMA prepared in this embodiment... 50 / SF 50 Semi-interpenetrating network hydrogels have high elasticity.

[0069] The biocompatibility of the hydrogels prepared in this embodiment and Comparative Examples 1-2 was tested. Human umbilical vein endothelial cells were seeded on the surface of the hydrogel and then cultured in a 37°C carbon dioxide (5%) incubator. The growth status of the cells on the hydrogel surface was photographed using laser confocal microscopy. Figure 8Figure (a) shows a confocal image of live / dead staining of cells cultured on day 7 using pure silk fibroin hydrogel on day 7; Figure (b) shows a confocal image of live / dead staining of cells cultured on day 7 using pure acylated chitosan hydrogel on day 7; Figure (c) shows a confocal image of CSMA staining. 50 / SF 50 The live / dead staining confocal image of cells cultured on day 7 in a semi-interpenetrating network hydrogel shows CSMA. 50 / SF 50 Compared to pure ultrasonic silk fibroin hydrogel and pure acylated chitosan hydrogel, CSMA exhibits greater cell proliferation on its surface, which suggests that CSMA... 50 / SF 50 Semi-interpenetrating network hydrogels exhibit good biocompatibility, which is superior to that of pure silk fibroin hydrogels and pure acylated chitosan hydrogels.

[0070] The hydrogel prepared in this embodiment has excellent biocompatibility and mechanical properties, and therefore can be used as a tissue surgical implant material in the field of biomedical materials.

[0071] Example 2

[0072] An artificial bone nail, the preparation method of which is as follows:

[0073] S1. Preparation of regenerated silk fibroin solution:

[0074] (1) Take 100g of silkworm silk, add 5000mL of deionized water, heat to boiling, add Na2CO3, continue to boil for 60min, then thoroughly wash the silkworm silk with deionized water, repeat this degumming process 3 times, the concentration of Na2CO3 added in the deionized water during the 3 degumming processes is 0.1wt%, 0.1wt%, and 0.05wt%, respectively, place the degummed silkworm silk in a 60℃ oven and dry for 24 hours, constantly loosening it during the process, and obtain the desired silk fibroin fiber after drying;

[0075] (2) Take 30g of silk fibroin and add it to 150mL of LiBr solution (9.3M). Place it in a 60℃ water bath and dissolve it for 30min. Then stir for 45min. Take out the solution, cool it, and transfer it to a dialysis bag. Dialyze it in a 4℃ refrigerator for 72h. Filter and centrifuge the dialyzed silk fibroin solution. Take the supernatant and dilute it to obtain a 5wt% regenerated silk fibroin solution. Then put it back into a dialysis bag and concentrate it in a 20wt% polyethylene glycol (number average molecular weight 2W) solution for 24h to obtain a 20wt% concentrated silk fibroin solution. Place it in a 4℃ refrigerator for later use.

[0076] S2. Preparation of acylated chitosan: 2g of chitosan (number average molecular weight 15W, degree of deacetylation 80-95%) was mixed with 98g of deionized water, placed in a 50℃ water bath and stirred for 60min, then 1.2mL of acetic acid solution (concentration 1.2% v / v) was added and stirred for 8h, and finally 7g of methacrylic anhydride was added and stirred for 4h. The reaction solution was taken out, cooled and placed in a 4℃ refrigerator for dialyzing for 72h. The dialyzed solution was filtered, centrifuged and poured into a glass dish and pre-cooled at -80℃ for 8h, and then freeze-dried in a freeze dryer for 60h to obtain acylated chitosan.

[0077] Preparation of S3, Highly Elastic Chitosan / Silk Fiber Semi-Interpenetrating Network Hydrogel: First, prepare a 0.135 wt% photoinitiator Irgacure 2959 solution. Disperse 0.2 g of the acylated chitosan obtained in S1 in 10 mL of the photoinitiator Irgacure 2959 solution, then add 10 mL of the concentrated silk fibroin solution prepared in S1. Stir evenly in the dark. The resulting blend solution is then ultrasonically treated with a power of 100 W for 3 seconds. After treatment, a bone screw mold is cast and immediately placed under a 600 W UV lamp for 10 minutes to cure the gel. This yields the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel (CSMA). 50 / SF 50 The semi-interpenetrating network hydrogel was frozen at -80°C for 10 minutes to remove it from the mold, then soaked in anhydrous alcohol for 48 hours, and finally dried in an oven at 37°C to obtain the artificial bone nail.

[0078] The artificial bone nail prepared in this embodiment is hard. To test the cell compatibility of the artificial bone nail prepared in this embodiment, osteoblasts were cultured on the surface of the bone nail (at 37°C in a 5% CO2 incubator). Laser confocal microscopy showed that the cells grew well on the artificial bone nail and exhibited a certain degree of adhesion. After culturing the osteoblasts on the bone nail surface for 3 days, the culture medium was removed with PBS, the cells were fixed, and then stained sequentially with phalloidin (to label the cytoskeleton) and DAPI (to label the cell nucleus). Cell growth was observed using a laser confocal microscope. Figure 9 For CSMA 50 / SF 50 Confocal images of Phalloidin / DAPI staining on day 3 of osteoblast culture seeded on a semi-interpenetrating hydrogel. From left to right: bright field image, DAPI staining under blue light excitation, Phalloidin staining under green light excitation, and staining under combined blue and green light excitation. It can be seen that CSMA... 50 / SF 50 The artificial bone screws prepared with hydrogels exhibited greater cell proliferation at the threaded sites, which suggests that CSMA...50 / SF 50 Semi-interpenetrating hydrogels have broad application prospects as artificial bone screws in bone repair.

[0079] In summary, this invention enables the preparation of CSMA / SF semi-interpenetrating network hydrogels with good resilience. After increasing the silk fibroin concentration and undergoing alcohol soaking treatment, the hydrogels exhibit a more rigid texture. Furthermore, the photocrosslinking hydrogel preparation process requires no additional catalysts for rapid gelation, allowing for cell seeding and culture on the hydrogel surface. Therefore, it shows promising application prospects in bone repair. The gel preparation process of this invention is simple, the gelation process is easily controlled, and the preparation cost is low, indicating a wide range of potential applications in the field of biomaterials.

[0080] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel, characterized in that, It is obtained by mixing a regenerated silk fibroin solution with a solution containing acylated chitosan, followed by ultrasonic treatment and photocuring. Its compressive stress is 1000~1200 kPa, maximum strain is 50~60%, and Young's modulus is 300~400 MPa. The concentration of the regenerated silk fibroin solution is 2.5~25wt%; the concentration of acylated chitosan in the acylated chitosan solution is 1~5wt%, and the acylated chitosan solution also contains a photoinitiator; the mass ratio of silk fibroin in the silk fibroin solution to acylated chitosan in the acylated chitosan solution is 1~10:1; The ultrasonic treatment process conditions are: ultrasonic power 100~200W, ultrasonic treatment time 3~60s; the photocuring conditions are: photocuring reaction is carried out under ultraviolet lamp irradiation, ultraviolet lamp power is 100~900W, and photocuring reaction time is 5~10min.

2. A method for preparing the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel according to claim 1, characterized in that, The specific steps are as follows: 1) Preparation of regenerated silk fibroin solution: Disperse the silk fibroin fibers obtained by degumming and drying silk in LiBr solution, heat and stir thoroughly, then dialyze. Filter and centrifuge the dialyzed silk fibroin solution, and dilute or concentrate as needed to obtain regenerated silk fibroin solution. 2) Preparation of acylated chitosan: Chitosan was added to deionized water and mixed well. The mixture was heated and stirred thoroughly. Then, acetic acid solution was added and stirred evenly. Finally, methacrylic anhydride was added and stirred for 3-6 hours. The reaction solution was taken out and dialyzed after cooling. The dialyzed solution was filtered, centrifuged and pre-cooled. Then, it was freeze-dried to obtain acylated chitosan. 3) Preparation of highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel: First, prepare a photoinitiator Irgacure 2959 solution, then disperse the acylated chitosan obtained in step 2) in the photoinitiator Irgacure 2959 solution to obtain a solution containing acylated chitosan, then mix it with the regenerated silk fibroin solution obtained in step 1) in proportion and stir evenly in the dark, then sonicate the resulting blend solution, and then place it under a UV lamp for light curing to obtain a highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel.

3. The method for preparing the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel according to claim 2, characterized in that, Step 1) The silk degumming method is as follows: add silk to deionized water, heat to boiling, add Na2CO3, continue boiling for 30-60 minutes, then thoroughly wash the silk with deionized water, repeat this process 2-3 times; Step 1) The concentration method is as follows: transfer the dilute regenerated silk fibroin solution obtained after centrifugation to a dialysis bag and soak it in a polyethylene glycol solution with a concentration of 20-30wt%.

4. The method for preparing the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel according to claim 2, characterized in that, Step 2) The chitosan has a number-average molecular weight of 10-20W, a degree of deacetylation of 80-95%, and a concentration of 1-4wt% in deionized water; Step 2) The acetic acid solution has a concentration of 1-3%, and the mass-to-volume ratio of chitosan to acetic acid solution is 1g / 0.24-1.5mL; Step 2) The mass ratio of methacrylic anhydride to chitosan is 1.4-8:1; Step 2) The pre-cooling conditions are: freezing at -60 to -80℃ for 8-12 hours.

5. The method for preparing the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel according to claim 2, characterized in that, In step 3), the concentration of the photoinitiator Irgacure 2959 solution is 0.1~0.5wt%; in step 3), the concentration of acylated chitosan in the acylated chitosan solution is 1~5wt%.

6. The application of the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel as a tissue surgical implant material and bone repair material in the field of biomedical materials.

7. The artificial bone screw further obtained from the highly elastic chitosan / silk fibroin semi-interpenetrating network hydrogel according to claim 1, characterized in that, The hydrogel is obtained by mixing a 15-25 wt% regenerated silk fibroin solution with an acylated chitosan solution, followed by ultrasonic treatment and photocuring. The hydrogel is then frozen, soaked in anhydrous ethanol for 48-72 hours, and finally dried.

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