A series of silk fibroin hydrogel materials with the same structure but different stiffness and their preparation processes

Abstract

This invention discloses a series of silk fibroin hydrogel materials with the same structure but different stiffnesses and their preparation process. First, degummed silk fibroin is used as raw material to prepare methacryloyl fibroin (SFMA) and silk fibroin nanofibers (BSNF), respectively. Then, SFMA and BSNF solutions are obtained. Finally, the SFMA, BSNF, and photoinitiator aqueous solutions are mixed uniformly to obtain a mixed solution. The mixed solution is adjusted to different pH values, and photocrosslinking is then performed. Compared to the shortcomings of current methods that control stiffness by hydrogel concentration, resulting in structural differences, this invention has a significant effect on controlling the stiffness of silk fibroin hydrogel materials. It can control the stiffness while ensuring consistency in hydrogel porosity and surface topology, and the prepared materials with different stiffnesses remain stable. This invention can be used in research and clinical applications related to the regulation of tissue repair by the mechanical microenvironment.

Description

Technical Field

[0001] This invention belongs to the field of tissue repair materials technology, and provides a series of silk fibroin hydrogel materials with the same structure but different stiffness and their preparation process. Background Technology

[0002] The mechanical microenvironment plays a significant regulatory role in tissue repair, and hydrogel materials are excellent media for regulating this microenvironment. However, existing studies generally adjust the mechanical properties of hydrogels by varying their concentrations or using different material mixing ratios, but this often results in corresponding changes to their structure. When applied to tissue repair research, this often involves alterations to hydrogel porosity, surface topology, and active components, making it difficult to achieve a single, consistent mechanical regulatory factor.

[0003] Silk fibroin (SF) is a natural bio-protein fiber extracted from silkworm silk. It boasts good biocompatibility and a wide range of tunable mechanical properties (Pa-MPa), making it widely used in wound repair research. Methacrylated silk fibroin (SFMA), due to its photocrosslinking properties, is extensively used in 3D printing and wound repair research. The stiffness of SFMA hydrogels can be controlled by adjusting the photoinitiated crosslinking intensity and time, as well as the catalyst concentration. However, hydrogels prepared from pure SFMA not only exhibit unstable mechanical properties, but also show inconsistent porosity and surface topology among SFMA hydrogels with different stiffnesses. Therefore, how to prepare silk fibroin hydrogels with identical structures but different stiffnesses is a key problem that needs to be solved for its application in tissue repair research. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a series of silk fibroin hydrogel materials with the same structure but different stiffness and their preparation process.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a preparation process for a series of silk fibroin hydrogel materials with the same structure but different stiffnesses, the specific steps of which are as follows:

[0007] (1) Degumming raw silk to obtain degummed silk fibroin;

[0008] (2) Preparation of methacrylamide silk fibroin SFMA: Degummed silk fibroin was dissolved in lithium bromide solution, glycidyl methacrylate (GMA) was added, and the reaction was carried out to obtain SFMA;

[0009] (3) Preparation of silk fibroin nanofiber solution, i.e., BSNF solution: Degummed silk fibroin is dissolved in lithium bromide solution.

[0010] Dialysis and centrifugation yielded an aqueous solution of silk fibroin, which was concentrated and self-assembled into metastable silk fibroin particles. The particles were then diluted with deionized water.

[0011] Incubation yields a BSNF solution;

[0012] (4) SFMA is prepared into an SFMA solution using deionized water. The SFMA solution, BSNF solution, and photoinitiator aqueous solution are mixed evenly to obtain a mixed solution. The mixed solution is adjusted to different pH values, poured into a mold, and subjected to photocrosslinking reaction. After post-treatment, the series of silk fibroin hydrogel materials are obtained. The pH value is 7.4 to 10.5. The photoinitiator aqueous solution is obtained by dissolving the photoinitiator in water.

[0013] Preferably, the specific method of step (1) is as follows: add 2.5g of raw silkworm silk to 1L of boiling 2.12g / L sodium carbonate solution, continue to boil and stir for 30-60 minutes, take it out and wash it with deionized water 3-5 times, and dry it in an oven at 60℃.

[0014] Preferably, in step (2), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and stirred to dissolve it to a concentration of 200 g / L. GMA is then added to make the concentration 424 mmol / L.

[0015] Preferably, in step (2), the reaction conditions are: 60℃ for 3 to 6 hours.

[0016] Preferably, in step (2), after the reaction is completed, the mixture is dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide and unreacted GMA. The mixture is then centrifuged 1 to 2 times to remove impurities and freeze-dried to obtain SFMA.

[0017] Further preferred centrifugation conditions are: 4℃, 9000rpm, 20 minutes.

[0018] Preferably, in step (3), the 9.3 mol / L lithium bromide solution is heated to 60°C, degummed silk fibroin is added, and the solution is stirred to dissolve it. The solution is then dialyzed in deionized water for 3 to 8 days using a 12-14 kDa dialysis membrane to remove lithium bromide. The solution is then centrifuged 1 to 2 times to remove impurities, resulting in a silk fibroin aqueous solution with a mass concentration of 4-6%.

[0019] Preferably, in step (3), the concentration is increased to 20% by mass at 60°C to obtain a concentrated solution, which is then self-assembled into metastable silk fibroin particles. The concentrated solution is then diluted to 2% by deionized water and incubated to obtain a BSNF solution.

[0020] Preferably, in step (3), the incubation conditions are: 60°C sealed incubation until a gel is formed.

[0021] Preferably, in step (4), the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

[0022] Preferably, in step (4), when preparing the mixture, the mass concentration of the SFMA solution is 10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the photoinitiator aqueous solution is 0.5%.

[0023] Preferably, in step (4), the volume ratio of SFMA solution, BSNF solution and photoinitiator aqueous solution is 1:1:1 when preparing the mixture.

[0024] Preferably, in step (4), the pH of the mixture is adjusted to 7.4 to 10.5, and more preferably 7.4, 8.9, 9.0, 9.2, 9.9, 10.5.

[0025] Preferably, in step (4), a 5% sodium hydroxide solution or a 99% triethanolamine solution is used for pH adjustment.

[0026] Preferably, in step (4), the photocrosslinking conditions are: 405nm light source, 10–60 mW / cm². 2 The time is 0.5 to 4 minutes.

[0027] Preferably, in step (4), the post-treatment method is to soak in phosphate buffer (2.0mM KH2PO4, 137mM NaCl, 10.0mM Na2HPO4, 2.7mM KCl, pH 7.4) for 12 to 24 hours.

[0028] The present invention also provides a series of silk fibroin hydrogel materials with the same structure but different stiffness, which are obtained by the aforementioned preparation process.

[0029] This invention also provides the application of the aforementioned series of silk fibroin hydrogel materials with the same structure but different stiffness in the preparation of tissue repair materials.

[0030] The beneficial effects of this invention are:

[0031] This invention provides a series of silk fibroin hydrogel materials with the same structure but different stiffnesses and their preparation process. First, degummed silk fibroin is used as raw material to prepare methacrylamide-treated silk fibroin and silk fibroin nanofibers, respectively. Then, SFMA and BSNF solutions are obtained. Finally, the SFMA, BSNF, and photoinitiator aqueous solutions are mixed uniformly to obtain a mixed solution. The mixed solution is adjusted to different pH values, and photocrosslinking is then performed. Compared to the shortcomings of current methods that control stiffness by hydrogel concentration, resulting in structural differences, this invention has a significant effect on controlling the stiffness of silk fibroin hydrogel materials. It can control the stiffness while ensuring consistency in hydrogel porosity and surface topology, and the prepared materials with different stiffnesses remain stable. This invention can be used in research and clinical applications related to the regulation of tissue repair by the mechanical microenvironment.

[0032] The existing silk fibroin hydrogel material prepared solely with 10% SFMA has a Young's modulus of 8.8 ± 0.5 kPa, but its mechanical properties are unstable, continuously increasing with standing time, reaching 484.0 ± 41.8 kPa by day 14. However, after modification by introducing BSNF into SFMA, the mechanical stability of the 10% SFMA / 2% BSNF mixed hydrogel material is significantly improved, with a Young's modulus of 139.3 ± 46.1 kPa, and this Young's modulus does not change with standing time (Table 1). To address the problem of altered porosity and surface topology caused by adjusting stiffness with different hydrogel concentrations, this invention obtains a series of hydrogels with different stiffnesses and the same porosity by adjusting the pH value of the same concentration of hydrogel. For example, adjusting the pH value of 10% SFMA / 2% BSNF to 7.4, 8.9, 9.0, 9.2, 9.9, and 10.5 yields a series of hydrogels with the same structure (…). Figure 2 The silk fibroin hydrogels with Young's moduli of 139.3±46.1kPa, 60.5±23.9kPa, 44.1±16.6kPa, 15.3±11.8kPa, 5.6±5.0kPa, and 1.5±0.9kPa respectively (Table 2) showed that their Young's moduli did not change significantly over time (Table 2), thus achieving the single mechanical control factor.

[0033] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein:

[0035] Figure 1 This diagram illustrates the preparation of mechanically stable silk fibroin hydrogel materials, comprising four parts: raw silk degumming process, SFMA preparation process, BSNF preparation process, and preparation process of mechanically stable SFMA / BSNF mixed silk fibroin hydrogels.

[0036] Figure 2 Scanning electron microscope images of 10% SFMA / 2% BSNF mixed hydrogels at different pH values. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] Example:

[0039] like Figure 1 As shown, the preparation process of a series of silk fibroin hydrogel materials with the same structure but different stiffnesses is as follows:

[0040] (1) Degumming of raw silk: Add 8.48g of sodium carbonate to 4L of deionized water and boil, stirring to dissolve it completely, to obtain a sodium carbonate solution with a concentration of 2.12g / L. Add 10g of raw silkworm silk and continue to boil and stir for 30 minutes. Wash with deionized water 5 times to remove sericin from the surface of the raw silk. Dry in an oven at 60℃ to obtain degummed silk fibroin.

[0041] (2) Preparation of SFMA: 20.19 g of lithium bromide was dissolved in 25 ml of deionized water and fully dissolved to obtain a lithium bromide solution with a concentration of 9.3 mol / L. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour to make the concentration of degummed silk fibroin 200 g / L. 1.5 ml of GMA was added to make the concentration of GMA 424 mmol / L. The reaction was carried out at 60 °C for 6 hours. The lithium bromide and unreacted GMA were removed by dialyzing with a 12 kDa dialysis membrane in deionized water for 7 days to obtain methacrylamide silk fibroin SFMA.

[0042] (3) Preparation of BSNF: 20.19 g of lithium bromide was dissolved in deionized water and fully dissolved to obtain a lithium bromide solution with a concentration of 9.3 mol / L. 5 g of degummed silk fibroin was added and dissolved at 60 °C for 1 hour. The solution was dialyzed with a 12 kDa dialysis membrane for 3 days to remove lithium bromide and centrifuged twice (4 °C, 9000 rpm, 20 min) to remove impurities, resulting in a silk fibroin aqueous solution with a concentration of 6 wt%. The solution was then slowly concentrated to 20% at 60 °C. The concentrated solution was diluted to 2% with deionized water and incubated in a sealed oven at 60 °C to obtain a BSNF solution with a mass concentration of 2%.

[0043] (4) Prepare an SFMA solution using deionized water. Mix a 10% SFMA solution, a 2% BSNF solution, and a 0.5% photoinitiator aqueous solution (obtained by dissolving lithium phenyl (2,4,6-trimethylbenzoyl) phosphate in water, or an LAP aqueous solution) at a volume ratio of 1:1:1 to obtain a homogeneous mixture. Adjust the pH of the mixture to different values ​​using triethanolamine. Pour the mixture into a 3cm dish and initiate the photocrosslinking reaction (405nm light source, 60mw / cm). 2 The photoinitiator was soaked in phosphate buffer (2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, 2.7 mM KCl, pH 7.4) for 12 hours to obtain the series of silk fibroin hydrogel materials described above; wherein, the photoinitiator aqueous solution was obtained by dissolving the photoinitiator lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP) in water.

[0044] Comparative example:

[0045] Omit BSNF in step (3), adjust the mass concentration of SFMA used in step (4) to 10% and the mass concentration of photoinitiator aqueous solution to 0.5% when preparing the mixture, and use triethanolamine to adjust the pH of the mixture to 7.4.

[0046] The rest is the same as in the embodiment.

[0047] The mechanical properties of the silk fibroin hydrogel materials obtained in the examples and comparative examples were tested using a bio-nanoindenter (Optics11, Netherlands), and the results are shown in Table 1.

[0048] Table 1 Comparison of mechanical properties of existing pure SFMA hydrogels and modified SFMA / BSNF hybrid hydrogels.

[0049]

[0050] As shown in Table 1, the Young's modulus of the silk fibroin hydrogel material prepared by simply using 10% SFMA was 8.8±0.5 kPa, but its mechanical properties were unstable and increased continuously with the standing time, reaching 484.0±41.8 kPa on the 14th day. However, after the 10% SFMA / 2% BSNF mixed hydrogel material was modified by introducing BSNF into SFMA, the mechanical stability of the 10% SFMA / 2% BSNF mixed hydrogel material was significantly improved, and its Young's modulus was 139.3±46.1 kPa, and the Young's modulus did not change with the standing time.

[0051] Selecting 10% SFMA / 2% BSNF from Table 1, that is, in step (4) of the example, when preparing the mixture, the mass concentration of the SFMA solution used is 10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the photoinitiator aqueous solution is 0.5%. Adjusting the pH of the mixture to 7.4, 8.9, 9.0, 9.2, 9.9, and 10.5 yields a series of structures with the same... Figure 2 The silk fibroin hydrogels with Young's moduli of 139.3±46.1kPa, 60.5±23.9kPa, 44.1±16.6kPa, 15.3±11.8kPa, 5.6±5.0kPa, and 1.5±0.9kPa respectively (Table 2) showed that their Young's moduli did not change significantly over time (Table 2), thus achieving the single mechanical control factor.

[0052] Table 2 Comparison of mechanical properties of 10% SFMA / 2% BSNF mixed hydrogels at different pH values

[0053]

[0054] Finally, it should be noted that 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 present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A process for the preparation of a series of silk fibroin hydrogel materials of the same structure but different stiffness, characterized in that, The specific steps are as follows: (1) degumming of raw silk to obtain degummed silk fibroin; (2) preparation of methacrylated silk fibroin SFMA: degummed silk fibroin is dissolved in lithium bromide solution, glycidyl methacrylate is added, and reaction is carried out to obtain SFMA; (3) preparation of silk fibroin nanofiber solution, i.e. BSNF solution: degummed silk fibroin is dissolved in lithium bromide solution, dialysis is carried out, centrifugation is carried out to obtain silk fibroin aqueous solution, concentration is carried out, metastable silk fibroin particles are self-assembled, deionized water is used for dilution, and incubation is carried out to obtain BSNF solution; (4) SFMA is prepared into SFMA solution with deionized water, SFMA solution, BSNF solution and aqueous solution of photoinitiator are uniformly mixed to obtain a mixed solution, the pH of the mixed solution is adjusted, the mixed solution is poured into a mold, photocrosslinking reaction is carried out, and post-treatment is carried out to obtain the series of silk fibroin hydrogel materials; wherein the pH is 7.4-10.5, and the aqueous solution of photoinitiator is obtained by dissolving the photoinitiator in water.

2. The manufacturing process according to claim 1, characterized in that, The specific method of step (1) is as follows: 2.5 g of domestic silkworm raw silk is added into 1 L of boiling 2.12 g / L sodium carbonate solution, boiling and stirring are continued for 30-60 minutes, and then the mixture is taken out and washed with deionized water for 3-5 times, and then dried in a 60℃ oven.

3. The manufacturing process of claim 1, wherein, In step (2), 9.3 mol / L lithium bromide solution is heated to 60℃, degummed silk fibroin is added, and stirring is carried out to dissolve the degummed silk fibroin, and the concentration is 200 g / L, and then GMA is continuously added to make the concentration of GMA be 424 mmol / L.

4. The manufacturing process of claim 1, wherein, In step (2), the reaction conditions are as follows: the reaction is carried out at 60℃ for 3-6 hours.

5. The manufacturing process of claim 1, wherein, In step (2), after the reaction is completed, 12-14 kDa dialysis membrane is used for dialysis in deionized water for 3-8 days to remove lithium bromide and unreacted GMA, and centrifugation is carried out for 1-2 times to remove impurities, and then freeze-drying is carried out to obtain SFMA.

6. The manufacturing process of claim 1, wherein, In step (3), 9.3 mol / L lithium bromide solution is heated to 60℃, degummed silk fibroin is added, and stirring is carried out to dissolve the degummed silk fibroin, and then 12-14 kDa dialysis membrane is used for dialysis in deionized water for 3-8 days to remove lithium bromide, and centrifugation is carried out for 1-2 times to remove impurities, and then a silk fibroin aqueous solution with a mass concentration of 4-6% is obtained.

7. The manufacturing process of claim 1, wherein, In step (3), the concentrated solution is obtained by concentrating the silk fibroin aqueous solution to a mass concentration of 20% at 60℃, and then metastable silk fibroin particles are self-assembled, and then the concentrated solution is diluted with deionized water to a mass concentration of 2% and incubated to obtain BSNF solution. The incubation conditions are as follows: incubation is carried out at 60℃ in a closed state until a gel-like substance is formed.

8. The manufacturing process of claim 1, wherein, In step (4), when the mixed solution is prepared, the mass concentration of the SFMA solution is 10%, the mass concentration of the BSNF solution is 2%, and the mass concentration of the aqueous solution of photoinitiator is 0.5%; the volume ratio of the SFMA solution, the BSNF solution and the aqueous solution of photoinitiator is 1:1:1; The pH of the mixed solution is adjusted to 7.4-10.5; Sodium hydroxide solution with a mass concentration of 5% or triethanolamine solution with a mass concentration of 99% is used for pH adjustment; The photo-crosslinking conditions are: 405 nm light source, 10-60 mw / cm 2 , time 0.5-4 minutes; The post-treatment method is as follows: the mixture is soaked in phosphate buffer solution for 12-24 hours.

9. A series of silk fibroin hydrogel materials of the same structure but different stiffness, characterized in that, The series of silk fibroin hydrogel materials are obtained by the preparation process of any one of claims 1-8.

10. Use of the series of silk fibroin hydrogel materials of the same structure and different stiffness according to claim 9 for preparing a tissue repair material.

Images