Preparation method of sericin hydrogel and application of sericin hydrogel obtained by the method

Abstract

The application discloses a preparation method of sericin hydrogel, which comprises the following steps: dissolving calcium ions in acid to prepare an acid solution of calcium ions; washing white silkworm cocoons without silk fibroin, drying, grinding into powder, and screening to obtain silkworm cocoon powder; taking the silkworm cocoon powder and the acid solution of calcium ions according to a mass-volume ratio of 0.01-0.8g:1mL, mixing uniformly, dissolving at 20-50 DEG C for 1-24h, and centrifuging at a rotating speed of 3000-15000rpm for 5-60min; collecting supernatant after centrifugation, pouring into a mold, and placing in an environment at 20-50 DEG C; after acid is fully volatilized, taking out a solid sample, and placing in ultrapure water for rinsing. The application further discloses the sericin hydrogel prepared by the method, and a sericin bone nail prepared by shaping the sericin hydrogel after drying treatment, and the application of the sericin hydrogel and the bone nail in soft tissue repair and hard tissue fixation respectively. The sericin hydrogel prepared by the method has excellent mechanical properties and transparency after being fully soaked in water, the tensile strength reaches 790-807kPa, and the elongation rate reaches 1100-1246%; after the sericin hydrogel is rinsed by ethanol and dried, the elastic modulus reaches 3.1GPa.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically a method for preparing sericin hydrogel, the resulting hydrogel, and its applications. Background Technology

[0002] Sericin is a natural protein extracted from silkworm cocoons, rich in 18 amino acids. It not only possesses excellent moisturizing, antioxidant, biodegradable, and swelling properties, but also exhibits good biocompatibility and low immunogenicity, supporting cell adhesion and proliferation and stimulating cell differentiation. Furthermore, sericin is rich in various functional groups, allowing it to undergo chemical modification and cross-linking. Its superior bioactivity and controllable physicochemical properties have garnered widespread attention and research in the field of biomedical materials.

[0003] Currently, sericin has been prepared into various forms of biomaterials, such as sericin scaffolds, sponges, hydrogels, conduits, and nanospheres. These materials have been applied to the repair of tissue damage in nerves, bones, muscles, and skin. Among them, sericin hydrogels, as a biomaterial whose composition and structure are very similar to the natural extracellular matrix, have attracted much attention. Although various sericin hydrogels have been prepared using different cross-linking methods, these sericin hydrogels suffer from problems such as low mechanical strength, poor ductility, low hardness, fragility, and poor transparency, which seriously restricts their application fields. Previously, the inventors' team prepared a relatively strong sericin hydrogel by extracting sericin from the silkworm's silk gland. However, the sericin used in this hydrogel was extracted from the silkworm's silk gland, which was time-consuming and labor-intensive, and the prepared sericin hydrogel lacked transparency. Therefore, developing a sericin hydrogel with high mechanical strength, high ductility, strength, and transparency with high cell compatibility remains a challenge in the development and application of new sericin materials.

[0004] Furthermore, due to their poor mechanical properties, there are currently no materials made from sericin for hard tissue fixation. How to prepare sericin materials with high mechanical strength is also a challenge in the field of new sericin materials. Summary of the Invention

[0005] Purpose of the invention: In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a simple method for preparing sericin hydrogel that does not require the introduction of crosslinking agents or polymers. Another purpose of this invention is to provide a sericin hydrogel with good biocompatibility and good tensile properties. Yet another purpose of this invention is to provide a role of sericin hydrogel in hard tissue fixation.

[0006] Technical solution: The present invention provides a method for preparing a sericin hydrogel, comprising the following steps:

[0007] (1) Dissolve calcium ions in acid to prepare an acid solution of calcium ions;

[0008] (2) Wash, dry, and grind white silkworm cocoons without silk fibroin into powder, and sieve to obtain silkworm cocoon powder;

[0009] (3) Weigh silkworm cocoon powder and calcium ion acid solution at a mass-volume ratio of 0.01-0.8g:1mL, mix well, place at 20-50℃ to dissolve for 1-24h, and centrifuge at 3000-15000rpm for 5-60min;

[0010] (4) After centrifugation, collect the supernatant, pour it into a mold, place it in an environment of 20-50℃, and after the acid has fully evaporated, take out the solid sample and rinse it in pure water to obtain sericin hydrogel.

[0011] Further, in step (1), the calcium ion is one or more of calcium chloride, calcium carbonate, calcium sulfate, and calcium hydroxide. The acid is one or more of formic acid, acetic acid, propionic acid, hydrochloric acid, and citric acid.

[0012] Furthermore, in step (2), the drying temperature is 10–50°C. The silkworm cocoon powder is sieved through a 10–300 mesh sieve. Compared to ordinary silkworm cocoons, silkworm cocoon powder does not contain fibroin and can be directly dissolved in a calcium chloride / calcium carbonate / calcium sulfate / calcium hydroxide-formic acid / acetic acid / propionic acid / hydrochloric acid / citric acid solution. This not only simplifies the process and increases the sericin extraction rate, but also results in a lower degradation rate of the obtained sericin protein.

[0013] Furthermore, in step (3), the dissolution time is preferably 4–8 h, which maximizes the dissolution rate of the silkworm cocoons and optimizes the hydrogel forming performance. The centrifugation speed is preferably 5000–15000 rpm, and the centrifugation time is preferably 15–40 min, with no significant change in the sericin content in the supernatant.

[0014] Furthermore, in step (4), the rinsing is performed 3 to 10 times, each time for 5 to 15 minutes.

[0015] The sericin hydrogel obtained by the above preparation method of the present invention, after being washed with ethanol and dried, has an elastic modulus of 2-4 GPa, a tensile strength of 750-807 kPa, and an elongation of 1100-1246%.

[0016] The application of the sericin hydrogel described in this invention in hard tissue fixation is specifically its application as a sericin bone screw.

[0017] Preparation principle: Silkworm cocoons are dissolved using an acidic solution of calcium ions. A new material with mechanical properties far exceeding those of previous pure sericin materials is prepared by multi-layer stacking, repeated drying, and rinsing with ethanol. This material has an elastic modulus as high as 3.1 GPa.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0019] 1. The obtained sericin hydrogel has excellent mechanical properties and transparency, with an elongation at break of up to 1246%, a maximum tensile strength of up to 807 kPa, and an elastic modulus of up to 2.3 GPa. In addition, the hydrogel also has good cell compatibility and can be used to prepare cartilage and tendon repair materials.

[0020] 2. The obtained hydrogel can be stacked layer by layer and dried to obtain a novel sericin material with an elastic modulus as high as 3.1 GPa. This material can be used to prepare sericin bone nails after molding treatment.

[0021] 2. Silkworm cocoon powder does not contain fibroin and can be directly dissolved in acidic solutions containing calcium ions. This not only makes the preparation process simple and results in a high sericin extraction rate, but also leads to a low degradation rate of the obtained sericin protein.

[0022] 3. For the first time, a solution of calcium chloride / calcium carbonate / calcium sulfate / calcium hydroxide-formic acid / acetic acid / propionic acid / hydrochloric acid / citric acid was used to dissolve silkworm cocoons to prepare hydrogel materials. This method is simple to operate, and hydrogels can be directly prepared after separating sericin without the need to introduce other cross-linking agents or polymers. Attached Figure Description

[0023] Figure 1 This is a flowchart of the preparation process of the present invention;

[0024] Figure 2 This is a microstructure diagram of the sericin hydrogel obtained in Example 1 of the present invention after low-temperature treatment and freeze-drying, wherein a is freeze-dried after treatment at -20℃, b is freeze-dried after treatment at -80℃, and c is freeze-dried after treatment at -196℃.

[0025] Figure 3 This is the infrared spectrum of the sericin hydrogel obtained in Example 1 of the present invention;

[0026] Figure 4 This is a data processing diagram of the infrared spectrum of the present invention;

[0027] Figure 5 This is the X-ray diffraction pattern of the sericin hydrogel obtained in Example 1 of the present invention;

[0028] Figure 6 This is a swelling rate curve of the sericin hydrogel obtained in Example 1 of the present invention under different pH conditions;

[0029] Figure 7 This is a graph showing the degradation rate of the sericin hydrogel obtained in Example 1 of the present invention under different pH conditions.

[0030] Figure 8 This is a schematic diagram of the mechanical properties of the sericin hydrogel obtained in Example 1 of the present invention;

[0031] Figure 9 These are microscopic images of the sericin hydrogel obtained in Example 1 of this invention and the control group co-cultured with cells at different culture times;

[0032] Figure 10 This is a schematic diagram illustrating the cell compatibility of the sericin hydrogel obtained in Example 1 of the present invention;

[0033] Figure 11 This is a flowchart illustrating the preparation process of the sericin bone nail of the present invention;

[0034] Figure 12 This is a graph showing the elastic modulus of the dried sericin bone nail prepared using Example 7. Detailed Implementation

[0035] In the following examples, calcium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd., anhydrous formic acid (AR, 98%) was purchased from Aladdin Company, and fibroin-deficient mutant silkworms were preserved and commercially available from the Sericulture Research Institute of the Chinese Academy of Agricultural Sciences. Silkworms with white cocoons lacking fibroin were selected and raised in a standard environment, fed fresh mulberry leaves throughout the process, with the cocooning temperature controlled at 25–27°C and humidity at 60%–70%; after spinning, the cocoons were collected.

[0036] Example 1

[0037] like Figure 1 A method for preparing a sericin hydrogel includes the following steps:

[0038] (1) Dissolve 0.4g of calcium chloride in 10mL of formic acid. After complete dissolution, dispense the calcium chloride-formic acid solution into 1.5mL EP tubes, with 1mL of calcium chloride-formic acid solution in each tube.

[0039] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 30°C. After they are dry, grind them into powder and pass them through a 100-mesh sieve to obtain silkworm cocoon powder for later use.

[0040] (3) Take 0.06g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 37℃ for 6h, and centrifuge at 12000rpm for 25min.

[0041] (4) After centrifugation, collect the supernatant, pour it into a mold, and place it in a 37℃ constant temperature incubator for 4 hours to fully volatilize the formic acid. Take out the solid sample and rinse it in pure water 3 times for 15 minutes each time to obtain the sericin hydrogel.

[0042] The performance of the sericin hydrogel obtained in Example 1 was tested, and the results are analyzed below:

[0043] a. Transparency of sericin hydrogel

[0044] A single-layer film of sericin hydrogel prepared by dissolving silkworm cocoons in a calcium chloride-formic acid solution exhibits good transparency.

[0045] b. Sericin hydrogel has a porous structure.

[0046] The sericin hydrogel obtained in Example 1 was frozen at -20℃, -80℃, and -196℃, respectively, and then freeze-dried in a low-temperature vacuum dryer. The structure was observed using a scanning electron microscope. Figure 2 As shown, the freeze-dried sericin hydrogel has a porous three-dimensional scaffold structure, and the pore size of the hydrogel scaffold decreases as the sample processing temperature decreases.

[0047] c. Porosity analysis of sericin hydrogels

[0048] The sericin hydrogel obtained in Example 1 was frozen at -20℃ and -80℃, respectively. After being freeze-dried in a low-temperature vacuum dryer, it was placed in pure water with a known volume of V1. The total volume at this point was recorded as V2. After standing at room temperature for 1 hour, the sericin hydrogel sample was taken out, and the remaining liquid volume was recorded as V3. The porosity of the sample is then:

[0049]

[0050] The porosities of the sericin hydrogels treated at -20℃ and -80℃ were 68% and 58%, respectively.

[0051] d. Infrared spectroscopy and X-ray diffraction analysis of sericin hydrogel

[0052] Fourier transform infrared spectroscopy (Nexus, Thermal Nicolet, USA) was used to determine the properties of sericin hydrogel at 4000-400 cm⁻¹. -1 Characteristic peaks. X-ray diffraction was performed using a rotating target X-ray diffractometer (D / MAX-RB, Japan, RIGAKU), with 2θ between 5 and 70°.

[0053] Figure 3 This is the FT-IR image of a sericin hydrogel. Figure 4The FT-IR data was processed by shearing, deconvolution, differentiation, peak separation, and curve fitting before plotting. Calculations showed that the proportions of β-sheets, random coils, α-helices, and β-turns were 43.59%, 22.41%, 0%, and 33.99%, respectively. Compared to hydrogels prepared from sericin isolated by lithium bromide (PolymerTesting 77(2019)105890, Polymer Testing 80(2019)106016), the sericin hydrogel in this invention has higher β-sheet and β-turn content, which is the reason for the superior mechanical properties of the sericin hydrogel in this invention.

[0054] like Figure 5 The sericin hydrogel exhibits two main diffraction peaks, at 11.45° and 19.57°. The 19.57° peak corresponds to β-sheet, indicating that the sericin hydrogel contains a large amount of β-sheet components. The 11.45° peak is a characteristic peak not previously reported in sericin hydrogels, suggesting that the sericin hydrogel in this invention differs from previously reported hydrogels. This may be an important factor contributing to the excellent mechanical properties of this sericin.

[0055] e. Swelling kinetics analysis of sericin hydrogel

[0056] After freeze-drying, the sericin hydrogel was weighed and recorded as M0. It was then immersed in PBS solutions with three different pH values ​​(pH 3.0, pH 7.4, pH 11.0) at 37°C. The PBS was replaced every two days. At specific time points, the sample was removed, surface moisture was removed, and the weight was recorded as M1. The swelling rate was calculated using the following formula:

[0057]

[0058] The results are as follows Figure 6 As shown, the swelling ratio of sericin hydrogel varies under different pH conditions, and the swelling effect is better under alkaline conditions than under neutral and acidic conditions.

[0059] f. Kinetic analysis of sericin hydrogel degradation

[0060] The degradation kinetics of sericin hydrogels were investigated under different pH conditions. Three samples were randomly selected from the same batch of sericin hydrogels, weighed, and recorded. After drying, they were weighed again to determine the wet-to-dry weight ratio of the batch. The samples used for degradation testing were weighed, and the initial sample weight was used to calculate the dry weight, denoted as Ma. These samples were then immersed in PBS (pH 3.0, 7.4, 11.0) at 37°C. The PBS was replaced every two days. At predetermined time points, the samples were removed, thoroughly rinsed, dried, and weighed, denoted as Mb. The degradation calculation formula for sericin hydrogels is as follows:

[0061]

[0062] from Figure 7 It can be seen that the sericin hydrogel degrades fastest in an alkaline environment (pH 11.0), with a cumulative degradation of 85% in 35 days; under acidic conditions, it degrades by 40% in 35 days; and under neutral conditions, it degrades by 31% in 35 days.

[0063] g. Mechanical property analysis of sericin hydrogel

[0064] Tensile tests were performed on the wetted hydrogel samples using a universal testing machine at a speed of 5 mm / min, and the tests were conducted at room temperature. The results are as follows: Figure 8 As shown, the sericin hydrogel prepared by the present invention using silkworm cocoons and calcium chloride formic acid solution has excellent mechanical properties, with a tensile strength of up to 807 kPa and an elongation of up to 1246%.

[0065] h. Cell compatibility analysis of sericin hydrogel

[0066] Sericin hydrogels were prepared in 24-well and 96-well plates, respectively, and then sterilized by immersion in 75% ethanol. After sterilization, they were rinsed with sterile distilled water and then soaked overnight in DMEM medium containing 10% fetal bovine serum. Mouse embryonic fibroblasts (NIH3T3) were cultured at 3.6 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1500 cells / well in the sericin hydrogel of 24-well plates and in blank control wells without hydrogel. Observations and photographs were taken daily using a DMI3000B inverted fluorescence microscope (Laica, Italy). Mouse embryonic fibroblasts (NIH3T3) were seeded at a density of 1500 cells / well in the sericin hydrogel of 96-well plates and in blank control wells without hydrogel. Cell viability was measured using the CCK-8 assay on the first and second days after seeding. All cells were cultured at 37°C in a 5% CO2 incubator, with three replicates per cell assay group.

[0067] like Figure 9As shown, cell growth on the sericin hydrogel was not significantly different from that of the control group, and cells on the sericin hydrogel could grow until day 23. Figure 10 CCK-8 assay results showed that there was no significant difference in cell growth on the sericin hydrogel and in the blank control well plate, indicating that the sericin hydrogel allows cell growth and has good biocompatibility.

[0068] like Figure 10 As shown, the elastic modulus diagram of the sericin bone screw material used in Example 7 is presented. It can be seen that the elastic modulus of the sericin bone screw material is 3.1 GPa, which is comparable to that of the regenerated silk fibroin bone screw.

[0069] application

[0070] like Figure 11 A method for preparing a sericin bone nail includes the following steps:

[0071] (1) Dissolve calcium chloride in formic acid to prepare a calcium chloride formic acid solution with a concentration of (5%, w / v), and then dispense the calcium chloride-formic acid solution into 1.5 mL EP tubes, with 1 mL of calcium chloride-formic acid solution in each tube;

[0072] (2) Wash the silkworm cocoons without fibroin deficiency three times with ultrapure water, then place them in a tray and dry them at 30°C. After they are dry, grind them into powder and pass them through a 250-mesh sieve to obtain silkworm cocoon powder for later use.

[0073] (3) Take 0.06g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 37℃ for 7h, and centrifuge at 12000rpm for 30min.

[0074] (4) After centrifugation, collect the supernatant and pour it into a mold containing a cylindrical empty tank (1 cm in diameter and 10 cm in height) with one open end. Add 1 mL of the supernatant and place the mold with the sealed end facing down in a 37°C incubator for 4 hours to allow for complete evaporation of formic acid. Then, add ultrapure water to the tank and rinse the sample three times for 15 minutes each time. Finally, place the mold containing the sample in an oven and dry it at 50°C.

[0075] (5) Add the supernatant containing sericin back into the dried mold tank containing the sample as in step (4) and proceed as in step 4.

[0076] (6) Repeat step (5) until a dry sericin hydrogel sample of ideal thickness is obtained;

[0077] (7) Take out the sample prepared in step (6) and shape it;

[0078] (8) After sizing the sample in step (7), rinse it with pure ethanol, take it out and dry it in an oven at a constant temperature of 50°C to obtain the sericin bone nail.

[0079] like Figure 12 The elastic modulus of the prepared sericin bone nail was 3 GPa.

[0080] Example 2

[0081] A method for preparing a sericin hydrogel includes the following steps:

[0082] (1) Dissolve 0.4g of calcium chloride in 10mL of formic acid. After complete dissolution, dispense the calcium chloride-formic acid solution into 1.5mL EP tubes, with 1mL of calcium chloride-formic acid solution in each tube.

[0083] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 30°C. After they are dry, grind them into powder and pass them through a 100-mesh sieve to obtain silkworm cocoon powder for later use.

[0084] (3) Take 0.06g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 37℃ for 6h, and centrifuge at 12000rpm for 25min.

[0085] (4) After centrifugation, collect the supernatant, pour it into a hydrogel cylindrical mold, place it in a 37℃ constant temperature incubator for 4 hours to fully volatilize the formic acid, take out the solid sample, rinse it in pure water 3 times for 15 minutes each time, and use a lathe to shape the initial hydrogel sample.

[0086] Example 3

[0087] A method for preparing a sericin hydrogel includes the following steps:

[0088] (1) Dissolve 0.4g of calcium carbonate in 10mL of acetic acid. After complete dissolution, dispense the calcium carbonate-acetic acid solution into 1.5mL EP tubes, with 1mL of calcium carbonate-acetic acid solution in each tube.

[0089] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 10°C. After they are dry, grind them into powder and pass them through a 50-mesh sieve to obtain silkworm cocoon powder for later use.

[0090] (3) Take 0.01g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 20℃ for 1h, and centrifuge at 3000rpm for 5min.

[0091] (4) After centrifugation, collect the supernatant, pour it into a mold, and place it in a 20℃ constant temperature incubator for 4 hours to fully evaporate the acetic acid. Take out the solid sample and rinse it in pure water 6 times for 5 minutes each time to obtain the sericin hydrogel.

[0092] The sericin hydrogel obtained in Example 2 was subjected to performance testing. The hydrogel had an elastic modulus as high as 3.5 GPa, indicating that the material has good hardness. After immersion in ultrapure water, the tensile strength reached 750 kPa and the elongation reached 1100%.

[0093] Example 4

[0094] A method for preparing a sericin hydrogel includes the following steps:

[0095] (1) Dissolve 0.4g of calcium sulfate in 10mL of propionic acid. After complete dissolution, dispense the calcium sulfate-propionic acid solution into 1.5mL EP tubes, with 1mL of calcium sulfate-propionic acid solution in each tube.

[0096] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 50°C. After they are dry, grind them into powder and pass them through a 200-mesh sieve to obtain silkworm cocoon powder for later use.

[0097] (3) Take 0.8g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 50℃ for 24h, and centrifuge at 15000rpm for 60min.

[0098] (4) After centrifugation, collect the supernatant, pour it into a mold, and place it in a 50℃ constant temperature incubator for 4 hours to fully volatilize the propionic acid. Take out the solid sample and rinse it in pure water 10 times for 10 minutes each time to obtain the sericin hydrogel.

[0099] The tensile strength of the sericin hydrogel obtained in this embodiment is 795 kPa, and the elongation is 1137%.

[0100] Example 5

[0101] A method for preparing a sericin hydrogel includes the following steps:

[0102] (1) Dissolve 0.2g calcium chloride and 0.2g calcium sulfate in 10mL hydrochloric acid. After complete dissolution, dispense the calcium ion hydrochloric acid solution into 1.5mL EP tubes, with 1mL of calcium ion hydrochloric acid solution in each tube.

[0103] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 20°C. After they are dry, grind them into powder and pass them through a 150-mesh sieve to obtain silkworm cocoon powder for later use.

[0104] (3) Take 0.2g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 30℃ for 4h, and centrifuge at 5000rpm for 15min.

[0105] (4) After centrifugation, collect the supernatant, pour it into a mold, place it in a 30℃ constant temperature incubator for 4 hours to fully evaporate the hydrochloric acid, take out the solid sample, rinse it in pure water 5 times, 8 minutes each time, and obtain the sericin hydrogel.

[0106] The tensile strength of the sericin hydrogel obtained in this embodiment is 773 kPa, and the elongation is 1235%.

[0107] Example 6

[0108] A method for preparing a sericin hydrogel includes the following steps:

[0109] (1) Dissolve 0.4g of calcium hydroxide in 10mL of citric acid. After complete dissolution, dispense the calcium hydroxide-citric acid solution into 1.5mL EP tubes, with 1mL of calcium hydroxide-citric acid solution in each tube.

[0110] (2) Wash the silkworm cocoons without silk fibroin deficiency three times. After washing, place them on a tray and dry them at 40°C. After they are dry, grind them into powder and pass them through a 100-mesh sieve to obtain silkworm cocoon powder for later use.

[0111] (3) Take 0.4g of silkworm cocoon powder from step (2), add it to the 1.5mL EP tube from step (1), mix well, dissolve at 40℃ for 8h, and centrifuge at 10000rpm for 40min.

[0112] (4) After centrifugation, collect the supernatant, pour it into a mold, and place it in a 40℃ constant temperature incubator for 4 hours to fully volatilize the citric acid. Take out the solid sample and rinse it in pure water 8 times for 12 minutes each time to obtain the sericin hydrogel.

[0113] The tensile strength of the sericin hydrogel obtained in this embodiment reaches 804 kPa, and the elongation reaches 1196%.

[0114] Example 7

[0115] The remaining steps in this embodiment are the same as in Embodiment 1, except that the dissolution time in step (3) is 1h, 4h, 8h, 12h, 16h, 20h, and 24h, respectively. Tests were conducted on the silkworm cocoon dissolution rate and gelling performance. The results showed that the dissolution time was 4–8h, with the highest dissolution rate and the best hydrogel gelling performance.

[0116] Example 8

[0117] The remaining steps in this embodiment are the same as in Embodiment 1, except that the centrifugation speeds in step (3) are 3000 rpm, 5000 rpm, 7000 rpm, 9000 rpm, 11000 rpm, 13000 rpm, and 15000 rpm. The sericin content in the supernatant was tested, and the results showed that the sericin content in the supernatant did not change significantly when the centrifugation speed was between 5000 and 15000 rpm.

[0118] Example 9

[0119] The remaining steps in this embodiment are the same as in Example 1, except that the centrifugation time in step (3) is 5 min, 15 min, 40 min, and 60 min, respectively. The sericin content in the supernatant was tested, and the results showed that the sericin content in the supernatant did not change significantly when the centrifugation time was 15 to 40 min.

[0120] Comparative Example 1

[0121] The remaining steps of this comparative example were the same as in Example 1, except that the silkworm cocoons without fibroin deficiency were replaced with those containing fibroin deficiency. The results showed that fibroin in the cocoons also dissolved, and fibroin and sericin were difficult to separate. The material prepared by combining fibroin and sericin exhibited strong immunogenicity.

Claims

1. A method for preparing a sericin hydrogel, characterized in that, Includes the following steps: (1) Dissolve calcium compounds in acid to prepare an acidic solution of calcium ions; (2) Wash the white silkworm cocoons that do not contain fibroin, dry them, grind them into powder, and sieve them to obtain silkworm cocoon powder; (3) Weigh silkworm cocoon powder and calcium ion acid solution at a mass-volume ratio of 0.01~0.8 g:1 mL, mix well, place at 20~50℃ to dissolve for 1~24 h, and centrifuge at 3000~15000 rpm for 5~60 min; (4) After centrifugation, collect the supernatant, pour it into a mold, place it in an environment of 20~50℃, and after the acid has fully evaporated, take out the solid sample and rinse it in ultrapure water to obtain the sericin hydrogel.

2. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (1), the calcium compound is one or more of calcium chloride, calcium carbonate, calcium sulfate, and calcium hydroxide.

3. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (1), the acid is one or more of formic acid, acetic acid, propionic acid, hydrochloric acid, and citric acid.

4. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (2), the drying temperature is 10~50℃.

5. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (2), the sieving is performed through a 10-300 mesh sieve.

6. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (3), the dissolution time is 4 to 8 hours.

7. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (3), the centrifugation speed is 5000-15000 rpm and the centrifugation time is 15-40 min.

8. The method for preparing a sericin hydrogel according to claim 1, characterized in that: In step (4), the rinsing is performed 3 to 10 times, each time for 5 to 15 minutes.

9. The sericin hydrogel obtained by the preparation method of the sericin hydrogel according to any one of claims 1 to 8, characterized in that: After being rinsed with ethanol and dried, the elastic modulus of the sericin hydrogel is 2-4 GPa; the tensile strength of the sericin hydrogel is 750-807 kPa, and the elongation is 1100-1246%.

10. The application of the sericin hydrogel according to claim 9 in the preparation of sericin bone nails.

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