Silk fibroin-based hydrogel double-layer composite membrane and preparation method

Abstract

The application discloses a silk fibroin-based hydrogel double-layer composite membrane and a preparation method thereof. The silk fibroin and double-bond modified hyaluronic acid are fully stirred and dissolved to prepare a base membrane with good barrier effect; the thiol-modified silk fibroin and the double-bond modified hyaluronic acid are uniformly blended, and a photoinitiator and a reducing agent are added, and under the condition of ultraviolet light, an interpenetrating network hydrogel can be prepared as an inner layer structure of a double-layer membrane. After freeze-drying and vacuum compounding, the silk fibroin hyaluronic acid dense fiber membrane serves as a dense layer of the double-layer composite membrane; and the silk fibroin hyaluronic acid hydrogel layer closely attached to the dense layer serves as a loose layer of the double-layer composite membrane. The double-layer composite membrane provided by the application has obvious dense layer and loose layer, good barrier effect and good mechanical performance.

Description

Technical Field

[0001] This invention relates to the field of biocomposite materials technology, and in particular to a silk fibroin-based hydrogel bilayer composite membrane with good barrier and mechanical properties, and its preparation method. Background Technology

[0002] Currently, with advancements in technology and the development of dental materials, dental implant surgery has become a routine treatment for missing or damaged teeth. Tooth loss or damage caused by periodontal disease, periapical inflammation, tumors, trauma, etc., often results in insufficient bone volume in the implant area, thus requiring artificial intervention. Commonly used clinical methods to achieve this include external bone grafting, vertical bone splitting, maxillary sinus lift, and guided bone regeneration (GBR). Among these, GBR is favored by implant dentists due to its minimally invasive nature. GBR uses a membrane material as a barrier to tightly cover the bone defect area, preventing surrounding connective tissue from invading the defect. A sufficiently strong GBR membrane prevents rapidly growing fibroblasts and connective tissue from ingrowing into the bone defect area, providing space and protection while promoting osteoblast formation by slower-growing osteoblasts. Furthermore, the barrier membrane protects the wound area from mechanical damage and saliva contamination. The membrane should also be selectively permeable to allow for the delivery of nutrients. In addition to being biocompatible, the membrane should ideally have good mechanical properties and biocompatibility.

[0003] Commercially available GBR membranes include polytetrafluoroethylene (PTFE), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), and collagen. However, most commercially available GBR membranes are non-degradable (requiring secondary removal, causing secondary harm to patients) or not rigid enough (unable to maintain bone space, causing bone space collapse), and cannot match the bone, which limits their application in bone regeneration. Summary of the Invention

[0004] In order to solve the above-mentioned technical problems, the present invention provides a silk fibroin-based hydrogel bilayer composite membrane and its preparation method.

[0005] In a first aspect, the present invention provides a silk fibroin-based hydrogel bilayer composite membrane, which is achieved by the following technical solution.

[0006] A silk fibroin-based hydrogel bilayer composite membrane, wherein the outer layer of the bilayer composite membrane is a silk fibroin-hyaluronic acid dense membrane and the inner layer is a silk fibroin-hyaluronic acid hydrogel.

[0007] Secondly, the present invention provides a method for preparing a silk fibroin-based hydrogel bilayer composite membrane, which is achieved by the following technical solution.

[0008] A method for preparing the above-mentioned silk fibroin-based hydrogel bilayer composite membrane includes the following steps:

[0009] S1. Dissolve unmodified silk fibroin in deionized water, with the amount of unmodified silk fibroin being 2-4 wt%. Add double-bonded modified hyaluronic acid, with the amount of double-bonded modified hyaluronic acid being 2-4 wt%. Stir thoroughly to dissolve and disperse evenly. Then, spread the solution onto a release membrane to form a base membrane. After drying, modify the base membrane using an ethanol vapor method. The modified base membrane serves as the outer layer structure of the bilayer membrane.

[0010] S2. Dissolve the thiol-modified silk fibroin in an aqueous solution containing the reducing agent tris(2-carbonylethyl) phosphate hydrochloride for 15-20 min, with the amount of thiol-modified silk fibroin being 5-20 wt%. Then add glycerol and mix thoroughly. Dissolve the double-bond-modified hyaluronic acid in an aqueous solution containing the photoinitiator LAP, with the amount of double-bond-modified hyaluronic acid being 1%-5%. Mix the thiol-modified silk fibroin solution and the double-bond-modified hyaluronic acid solution thoroughly and set aside.

[0011] S3. Adhere a 1mm thick silicone mold to the base film obtained in step S1, pour the silk fibroin / hyaluronic acid hydrogel precursor solution obtained in step S2 evenly into it, irradiate the adhesive layer with ultraviolet light, and after freeze-drying and vacuum lamination, obtain a silk fibroin-based hydrogel bilayer composite film.

[0012] The present invention can regulate the mechanical properties of hydrogels by controlling the content of thiol-modified silk fibroin, and regulate the flexibility of hydrogels by changing the content of glycerol.

[0013] Furthermore, the preparation methods for modified or unmodified silk fibroin are as follows:

[0014] Preparation of degummed silk: Weigh 25-35g of shredded silkworm cocoons and boil them in 5-7L of 0.05M sodium carbonate aqueous solution for 30-50 minutes. Repeat the above steps 3-4 times. Wash the boiled silkworm cocoons with deionized water 4-5 times and dry them overnight at 55-70℃ to obtain degummed silk.

[0015] Preparation of silk fibroin solution: Immerse 5.0-5.4g of degummed silk in 70-80ml of CaCl2-C2H60-H2O solution, heat and stir at 65-75℃ for 3.5-4h; after dissolution, remove and cool to room temperature, centrifuge at 8000-10000rpm for 5-7min to remove insoluble impurities, and dialyze in a 3500-5000MWCO dialysis bag at 3-4℃ for 3-4 days;

[0016] Preparation of unmodified silk fibroin: After freeze-drying the silk fibroin solution, unmodified silk fibroin was obtained;

[0017] Preparation of thiol-modified silk fibroin: The silk fibroin solution in the dialysis bag was adjusted with 0.1-0.2M MES buffer at pH 6-6.5 for 20-24 h; after the pH value stabilized, 0.2-0.4 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4-0.6 mol / L N-hydroxysuccinimide were added to activate the reaction for 15-30 min, followed by the addition of 0.18-0.2 mol / L reduced glutathione and reaction at room temperature for 18-24 h. Dialysis was performed at 3-4 °C for 2-3 days, and the thiol-modified silk fibroin was obtained by freeze drying.

[0018] The thiol-modified silk fibroin described in this application is prepared by amide condensation reaction of silk fibroin molecules and reduced glutathione. The reduced glutathione grafted onto the thiol-modified silk fibroin is easily oxidized to oxidized glutathione, requiring the addition of a reducing agent to open the disulfide bonds before hydrogel preparation. The thiol-modified silk fibroin is a highly water-soluble protein.

[0019] Further, the preparation method of double-bond modified hyaluronic acid is as follows: Sodium hyaluronate with a molecular weight of 100,000 is dissolved in deionized water to a final concentration of 2-3 wt%. After stirring and dissolving, DMF is added at a volume ratio of DI:DMF = 3:2. After stirring and mixing, the mixture is placed in a condensing circulation system at 3-4℃ and cooled for 20-30 min. After the temperature stabilizes, 2.5-3 ml of methacrylic anhydride is added dropwise, and the reaction is allowed to proceed for 20-30 min. The pH value is adjusted to 8-9 with sodium hydroxide solution, and the reaction is allowed to proceed for 20-24 h. Then, 0.1-0.2 M sodium chloride solid is added and the reaction is allowed to proceed for 0.5-1 h. The solid is extracted by centrifugation using 2.5-3 times the volume of anhydrous ethanol as a precipitant. After redissolving in deionized water, the solid is dialyzed for 2-3 days and then freeze-dried to obtain double-bond modified hyaluronic acid.

[0020] Furthermore, the amount of tris(2-carbonylethyl)phosphohydrochloride used is 0.15–0.20 wt%.

[0021] Furthermore, the amount of glycerin used is 0.5-4 wt%.

[0022] Furthermore, the amount of photoinitiator LAP used is 0.1–0.5 wt%.

[0023] This invention utilizes SF (silk fibroin) extracted from silkworm cocoons, which possesses excellent biocompatibility and mechanical properties, as a matrix material. This matrix is ​​uniformly blended with double-bond modified hyaluronic acid (HHA) and coated to create a dense silk fibroin-hyaluronic acid membrane, serving as the outer layer of a bilayer composite membrane. Thiol-modified silk fibroin and double-bond modified hyaluronic acid are then uniformly blended, and glycerol solution, photoinitiator, and reducing agent are added. Under ultraviolet light, an interpenetrating hydrogel network is prepared, serving as the inner layer of the bilayer membrane. A porous silk fibroin-based hydrogel scaffold is then freeze-dried in situ on top of the dense silk fibroin-hyaluronic acid membrane, resulting in a novel functional bilayer GBR membrane. On one hand, the dense silk fibroin-hyaluronic acid layer, as the outer layer, is directly adjacent to the connective tissue and gingival tissue in the oral cavity. The dense membrane pores can prevent fibroblasts from ingrowing into bone defect areas. On the other hand, the silk fibroin-hyaluronic acid porous hydrogel scaffold, serving as the inner layer, provides the material with excellent mechanical properties, supports the upper connective tissue, prevents connective tissue collapse, and provides growth space for tissue regeneration; simultaneously, it mimics the porous morphology of bone trabeculae, directly contacting the bone defect area. This unique bilayer composite membrane structure is designed to adapt to the histological complexity between oral soft and hard tissues.

[0024] This application has the following beneficial effects.

[0025] 1. The silk fibroin-based hydrogel bilayer composite membrane of the present invention has an outer layer of silk fibroin-hyaluronic acid dense membrane and an inner layer of silk fibroin-hyaluronic acid porous hydrogel. The raw materials are natural and non-toxic, biodegradable, biocompatible, readily available, and low in cost.

[0026] 2. The method for preparing the silk fibroin-based hydrogel bilayer composite membrane of the present invention is simple to operate, adjustable, has good repeatability, and produces a stable finished product;

[0027] 3. In the silk fibroin-based hydrogel bilayer composite membrane of the present invention, the outer silk fibroin hyaluronic acid dense membrane effectively isolates fibroblast ingrowth, provides space for bone defect areas and plays a protective role, while the inner layer is a silk fibroin-based hydrogel scaffold layer with a honeycomb-like porous structure that facilitates cell ingrowth. This bilayer composite membrane structure effectively mimics the bone structure, has a biomimetic effect, effectively promotes bone tissue regeneration, and is expected to be applied to GBR.

[0028] 4. In the silk fibroin-based hydrogel bilayer composite membrane of the present invention, the outer silk fibroin hyaluronic acid dense membrane can inhibit and isolate fibroblast ingrowth, which is beneficial to maintaining the time and space for new bone formation in the bone defect area, and is more conducive to bone integration around the implant and bone regeneration in the defect area. Attached Figure Description

[0029] Figure 1This is a flowchart of the preparation process of the inner silk fibroin-based hydrogel membrane of the present invention (a: reaction of thiol modification of silk fibroin (SF-GSH); b: reaction of double bond modification of hyaluronic acid (HAMA); c: preparation of silk fibroin / hyaluronic acid hydrogel with interpenetrating network).

[0030] Figure 2 This is a scanning electron microscope (SEM) image of the silk fibroin-based hydrogel bilayer composite membrane prepared in Example 1 of the present invention (a: outer layer: silk fibroin hyaluronic acid layer; b: silk fibroin hyaluronic acid porous hydrogel layer). Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0032] Example 1:

[0033] A method for preparing a silk fibroin-based hydrogel bilayer composite membrane, the method comprising the following steps:

[0034] (1) Degummed silk: Weigh 30 pieces of shredded silkworm cocoons and boil them in 6L of 0.05M sodium carbonate aqueous solution for 40 minutes. Repeat the above steps three times. Wash the boiled silkworm cocoons 4 times with deionized water and dry them overnight in an electric hot air drying oven at 60℃ to obtain degummed silk.

[0035] Preparation of silk fibroin solution: 5g of degummed silk was immersed in approximately 75ml of CaCl2-C2H60-H2O solution and heated and stirred at 70℃ for 4h. After dissolution, the reaction flask was removed and cooled to room temperature. Insoluble impurities were removed by centrifugation at 8000rpm (6min, room temperature). The solution was then dialyzed in a MWCO3500 dialysis bag at 4℃ for 3 days.

[0036] Preparation of unmodified silk fibroin: A portion of the sample was directly freeze-dried in a freeze dryer to obtain a white, soft solid, which is unmodified silk fibroin.

[0037] Preparation of thiol-modified silk fibroin: After dialysis of the remaining sample, the silk fibroin solution in the dialysis bag was adjusted for 24 h with 0.2 M MES buffer (containing 0.05 M sodium chloride) at pH 6. After the pH stabilized, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.4 mol / L) and N-hydroxysuccinimide (0.6 mol / L) were added to activate the reaction for 30 min. Then, reduced glutathione (0.2 mol / L) was added and reacted at room temperature for 24 h. Dialysis was performed at 4 °C for 3 days, and freeze-drying yielded a white, dense solid, which was the thiol-modified silk fibroin (SF-GSH).

[0038] (2) Preparation of double-bond modified hyaluronic acid: Sodium hyaluronate with a molecular weight of 100,000 was dissolved in deionized water (2wt%) and stirred until a colorless and transparent solution was obtained. DMF was added at a volume ratio of DI:DMF = 3:2. After mixing, the solution was placed in a condenser circulation system at 4℃ and cooled for 30 min. After the temperature stabilized, 3 ml of methacrylic anhydride was slowly added dropwise, and the reaction was allowed to proceed for 30 min. The pH was adjusted to 8.5 with sodium hydroxide solution (1 mol / L). After reacting for 24 h, sodium chloride solid (0.2 M) was added and reacted for 1 h. A white flocculent precipitate was obtained using 3 times the volume of anhydrous ethanol as a precipitant. The solid was extracted by centrifugation, redissolved in deionized water, dialyzed for 3 days, and freeze-dried to obtain a white spongy solid, which is double-bond modified hyaluronic acid (HAMA).

[0039] (3) Preparation of the outer layer structure of the bilayer membrane: Take the unmodified silk fibroin obtained in step (1) and dissolve it in deionized water (2wt%). Add the HAMA obtained in step (2) (2wt%), stir and dissolve thoroughly, disperse evenly, and then use a coating machine to form a base film on the release membrane. After natural drying, modify the base film by ethanol vapor method. The modified base film serves as the outer layer structure of the bilayer membrane.

[0040] (4) Dissolve the thiol-modified silk fibroin (9wt%) obtained in step (1) in an aqueous solution containing the reducing agent tris(2-carbonylethyl) phosphate hydrochloride (0.20wt%) for 15 min, then add glycerol (2wt%) and mix them evenly; dissolve the double bond-modified hyaluronic acid (1.8wt%) obtained in step (2) in an aqueous solution containing the photoinitiator LAP (0.5wt%); mix the two solutions evenly and set aside for later use.

[0041] (5) Preparation of bilayer composite film: A silicone mold with a thickness of 1 mm is adhered to the base film obtained in step (3), and the silk fibroin / hyaluronic acid hydrogel obtained in step (4) is poured into it evenly. The adhesive layer is irradiated with ultraviolet light (365 nm), and finally it is placed in a freeze dryer at -50°C for vacuum freeze drying for 12 h. After freeze drying and vacuum composite, the silk fibroin protein-based hydrogel bilayer composite film is finally obtained.

[0042] Performance testing

[0043] The thickness was measured using vernier calipers, and the membrane was prepared to measure 10×20×0.2mm. The mechanical properties of the membrane were tested using an electronic universal testing machine. Sandpaper was used to clamp both ends of the sample to prevent slippage during the test. The speed was set to 1mm / min. The yield point value of the stress-strain curve was used as the tensile strength, and the elastic modulus was calculated using the slope of the linear portion of the curve.

[0044] Table 1 Mechanical strength of the bilayer composite membrane under dry and wet conditions

[0045]

[0046] Table 2 Mechanical strength of commercial collagen membranes (Yiling) in dry and wet states.

[0047]

[0048] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A silk fibroin-based hydrogel double-layer composite membrane, characterized in that: The outer layer of the double-layer composite membrane is a dense membrane of silk fibroin and hyaluronic acid, and the inner layer is a hydrogel of silk fibroin and hyaluronic acid. The method for preparing the silk fibroin-based hydrogel bilayer composite membrane includes the following steps: S1. Dissolve unmodified silk fibroin in deionized water, with the amount of unmodified silk fibroin being 2-4 wt%. Add double-bonded modified hyaluronic acid, with the amount of double-bonded modified hyaluronic acid being 2-4 wt%. Stir thoroughly to dissolve and disperse evenly. Then, spread the solution onto a release membrane to form a base membrane. After drying, modify the base membrane using an ethanol vapor method. The modified base membrane serves as the outer layer structure of the bilayer membrane. S2. Dissolve the thiol-modified silk fibroin in an aqueous solution containing the reducing agent tris(2-carbonylethyl) phosphate hydrochloride for 15–20 min, with the amount of thiol-modified silk fibroin being 5–20 wt%. Then add glycerol and mix thoroughly. Dissolve the double-bond-modified hyaluronic acid in an aqueous solution containing the photoinitiator LAP, with the amount of double-bond-modified hyaluronic acid being 1%–5%. Mix the thiol-modified silk fibroin solution and the double-bond-modified hyaluronic acid solution thoroughly and set aside. S3. Adhere a 1mm thick silicone mold to the base film obtained in step S1, pour the silk fibroin / hyaluronic acid hydrogel precursor solution obtained in step S2 evenly into it, irradiate the adhesive layer with ultraviolet light, and after freeze-drying and vacuum lamination, obtain a silk fibroin-based hydrogel bilayer composite film.

2. A method for preparing the silk fibroin-based hydrogel bilayer composite membrane according to claim 1, characterized in that: Includes the following steps: S1. Dissolve unmodified silk fibroin in deionized water, with the amount of unmodified silk fibroin being 2-4 wt%. Add double-bonded modified hyaluronic acid, with the amount of double-bonded modified hyaluronic acid being 2-4 wt%. Stir thoroughly to dissolve and disperse evenly. Then, spread the solution onto a release membrane to form a base membrane. After drying, modify the base membrane using an ethanol vapor method. The modified base membrane serves as the outer layer structure of the bilayer membrane. S2. Dissolve the thiol-modified silk fibroin in an aqueous solution containing the reducing agent tris(2-carbonylethyl) phosphate hydrochloride for 15–20 min, with the amount of thiol-modified silk fibroin being 5–20 wt%. Then add glycerol and mix thoroughly. Dissolve the double-bond-modified hyaluronic acid in an aqueous solution containing the photoinitiator LAP, with the amount of double-bond-modified hyaluronic acid being 1%–5%. Mix the thiol-modified silk fibroin solution and the double-bond-modified hyaluronic acid solution thoroughly and set aside. S3. Adhere a 1mm thick silicone mold to the base film obtained in step S1, pour the silk fibroin / hyaluronic acid hydrogel precursor solution obtained in step S2 evenly into it, irradiate the adhesive layer with ultraviolet light, and after freeze-drying and vacuum lamination, obtain a silk fibroin-based hydrogel bilayer composite film.

3. The method for preparing a silk fibroin-based hydrogel bilayer composite membrane according to claim 2, characterized in that: The preparation methods for modified or unmodified silk fibroin are as follows: Preparation of degummed silk: Weigh out the shredded silkworm cocoons and boil them in sodium carbonate aqueous solution for 30-50 minutes. Repeat the above steps 3-4 times. Wash the boiled silkworm cocoons 4-5 times with deionized water and dry them overnight at 55-70℃ to obtain degummed silk. Preparation of silk fibroin solution: Degummed silk is immersed in CaCl2-C2H60-H2O solution and heated and stirred at 65-75℃ for 3.5-4h; after dissolution, it is taken out and cooled to room temperature, centrifuged at 8000-10000rpm for 5-7min to remove insoluble impurities, and dialyzed in a dialysis bag with MWCO3500-5000 at 3-4℃ for 3-4 days; Preparation of unmodified silk fibroin: After freeze-drying the silk fibroin solution, unmodified silk fibroin was obtained; Preparation of thiol-modified silk fibroin: The silk fibroin solution in the dialysis bag was adjusted with 0.1-0.2M MES buffer at pH 6-6.5 for 20-24 h; after the pH value stabilized, 0.2-0.4 mol / L 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4-0.6 mol / L N-hydroxysuccinimide were added to activate the reaction for 15-30 min, followed by the addition of 0.18-0.2 mol / L reduced glutathione and reaction at room temperature for 18-24 h. Dialysis was performed at 3-4 °C for 2-3 days, and the thiol-modified silk fibroin was obtained by freeze drying.

4. The method for preparing a silk fibroin-based hydrogel bilayer composite membrane according to claim 2, characterized in that: The preparation method of double-bond modified hyaluronic acid is as follows: Sodium hyaluronate with a molecular weight of 100,000 is dissolved in deionized water to a final concentration of 2-3 wt%. After stirring and dissolving, DMF is added at a volume ratio of DI:DMF = 3:

2. After stirring and mixing, the mixture is placed in a condensing circulation system at 3-4℃ and cooled for 20-30 min. After the temperature stabilizes, 2.5-3 ml of methacrylic anhydride is added dropwise, and the reaction is allowed to proceed for 20-30 min. The pH value is adjusted to 8-9 with sodium hydroxide solution, and the reaction is allowed to proceed for 20-24 h. Then, 0.1-0.2 M sodium chloride solid is added and the reaction is allowed to proceed for 0.5-1 h. The solid is extracted by centrifugation using 2.5-3 times the volume of anhydrous ethanol as a precipitant. After redissolving in deionized water, the solid is dialyzed for 2-3 days and then freeze-dried to obtain double-bond modified hyaluronic acid.

5. The method for preparing a silk fibroin-based hydrogel bilayer composite membrane according to claim 2, characterized in that: The amount of tri(2-carbonylethyl)phosphohydrochloride used is 0.15 to 0.20 wt%.

6. The method for preparing a silk fibroin-based hydrogel bilayer composite membrane according to claim 2, characterized in that: The amount of glycerin used is 0.5-4 wt%.

7. The method for preparing a silk fibroin-based hydrogel bilayer composite membrane according to claim 2, characterized in that: The amount of photoinitiator LAP used is 0.1–0.5 wt%.

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