Injectable fibroin piezoelectric hydrogel

Abstract

The injectable silk fibroin piezoelectric hydrogel comprises PVDF (Polyvinylidene Fluoride) spinning and regenerated silk protein with methacrylic anhydride groups, and the PVDF spinning and the regenerated silk protein are cross-linked to form a network structure. The invention also discloses a preparation method of the injectable silk fibroin piezoelectric hydrogel. The injectable silk fibroin piezoelectric hydrogel is applied to a bone injury part, can generate electrical stimulation under a load for bone repair, and has a clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and tissue regeneration, and relates to an injectable silk fibroin piezoelectric hydrogel and its use in bone repair. Background Art

[0002] Bone defect refers to bone shortage beyond a certain size caused by various reasons, and the defect that the body cannot heal itself by bone formation. Autologous bone grafting is currently the gold standard for treating bone defects, but factors such as secondary surgery and donor site trauma limit its clinical application. Therefore, constructing a bone repair material similar to natural bone in function, composition and structure is an effective method to solve the difficult problem of bone defect treatment.

[0003] In terms of bone repair, piezoelectric materials have shown great potential in tissue engineering and regeneration. Bones themselves also have piezoelectric properties, and the charge / potential generated in response to mechanical activity can promote bone growth. Piezoelectric materials can deform with physiological movements, thereby providing electrical stimulation to cells or damaged tissues without the need for an external power source. At the same time, piezoelectric materials can also stimulate the physiological electrical microenvironment, promote the proliferation and differentiation of stem cells to affect the bone regeneration process, and play a crucial role in regeneration and repair. Due to their ability to generate charge / potential in response to mechanical deformation, piezoelectric materials show great potential in manufacturing intelligent stimulation scaffolds for bone tissue engineering. For example, the inventor disclosed a silk protein / nano-zinc oxide composite piezoelectric hydrogel for bone repair in the patent document with the publication number CN115970055A.

[0004] Piezoelectric materials have more advantages in the repair of load-bearing bone tissues because physiological movements can induce such materials to generate electrical stimulation, regulating the electrophysiological microenvironment of bone tissues to promote the growth of bone cells. In addition, piezoelectric materials are also more attractive due to their antibacterial activity. Because severe bone injuries are often accompanied by factors such as infection and inflammatory microenvironment, anti-inflammatory treatment is required to improve the efficiency of new bone formation. The charge spontaneously generated by piezoelectric materials interacts with the charges of tissue cells, which can increase the permeability of inflammatory factors and exert anti-inflammatory efficacy. Piezoelectric materials can also promote the polarization of macrophages from inflammation to anti-inflammatory phenotype by regulating the immune microenvironment at the bone defect site, and synergistically promote bone healing and bone regeneration by regulating the immune microenvironment.

[0005] When the silk fibroin-based piezoelectric hydrogel of the prior art is used clinically, it is necessary to determine the bone injury site through imaging scans, estimate the shape and three-dimensional size of the bone defect, and then pour the hydrogel formulation solution, that is, the gel precursor solution, into a mold with a preset shape consistent with the bone defect shape. It is left standing at room temperature for a long time (heating if necessary) until a gel is formed, and then the gel is soaked in an ethanol solution for a quite long time to obtain a gel mold that can be implanted into the body. Then, through surgery, the molded gel mold is placed at the bone defect site. The whole process takes a long time, even several days; moreover, the molded gel mold may not be completely consistent with the shape and size of the bone defect, which may lead to surgical failure. Summary of the Invention

[0006] In order to overcome the above-mentioned defects of the silk fibroin-based piezoelectric materials for bone repair in clinical use in the prior art, the inventors tried to develop a silk fibroin-based piezoelectric material that can inject a bone-forming gel into the bone defect site in the form of injection. After a large number of experimental explorations, a bone-forming material in injection form with high biocompatibility, biodegradable / absorbable by the body, and having piezoelectric effect at the same time was finally developed. The gel-forming speed is fast, it can be shaped immediately during bone repair surgery, and the surgical success rate is significantly improved, greatly reducing the harm caused to the recipient by surgery. Specifically, the present invention includes the following technical solutions.

[0007] An injectable silk fibroin piezoelectric hydrogel, especially an injectable silk fibroin piezoelectric hydrogel for in vivo bone repair, characterized in that it includes PVDF (polyvinylidene difluoride) spinning and regenerated silk fibroin (SF-MA) with methacrylic anhydride groups (MA), and the two crosslink to form a network structure.

[0008] Before the crosslinking of the PVDF spinning and the regenerated silk fibroin (SF-MA) with methacrylic anhydride groups, they already have network structures respectively.

[0009] The nanoscale refers to dimensions in the range of 10 - 400 nm, such as 20 - 350 nm, 30 - 300 nm, 40 - 250 nm, 50 - 200 nm.

[0010] Furthermore, the above PVDF is a biocompatible polyvinylidene difluoride that can be biodegradable / absorbed by the body.

[0011] In one embodiment, the content of the above PVDF in the hydrogel is 5 - 15 mg / ml.

[0012] The above-mentioned regenerated silk fibroin with methacrylic anhydride groups (SF-MA) is prepared by reacting the stock solution of regenerated silk fibroin (RSF) with glycidyl methacrylate (GMA).

[0013] Preferably, the raw materials for preparing the injectable silk fibroin piezoelectric hydrogel further include triethanolamine (TEA), N-vinyl-2-pyrrolidinone (NVP), and a photo-crosslinking agent L2959 (photoinitiator 2959, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone).

[0014] The piezoelectric properties of the above-mentioned injectable silk fibroin piezoelectric hydrogel are as follows: The mechanical-electrical response signal is measured by a linkage device of an ultrasonic therapeutic instrument and a KEITHLEY DMM6500, generating a voltage of more than 0.03V, preferably more than 0.04V, and more preferably more than 0.05V.

[0015] In one embodiment, the above-mentioned regenerated silk fibroin with methacrylic anhydride groups (SF-MA) can be prepared by a method including the following steps:

[0016] 1) Degum the natural mulberry cocoons, dry them to make degummed silk, dissolve the degummed silk, and prepare a stock solution of regenerated silk fibroin (RSF).

[0017] 2) Mix the stock solution of regenerated silk fibroin (RSF) obtained in step 1) with glycidyl methacrylate (GMA), react to obtain an SF-MA solution, and perform dialysis and freeze-drying to obtain SF-MA powder.

[0018] Optionally, the operation mode of the above step 1) is as follows:

[0019] 1-1) Immerse the mulberry cocoons in a soap salt solution for degumming and drying to make degummed silk, and the soap salt is sodium carbonate or sodium bicarbonate.

[0020] Further, the soap salt in the above step 1-1) is sodium carbonate, and the concentration of the sodium carbonate solution is 1-10 wt%.

[0021] 1-2) Dissolve, heat, and dialyze the degummed silk obtained in step 1-1) with a lithium bromide solution to obtain a stock solution of regenerated silk fibroin.

[0022] Further, the concentration of the lithium bromide solution in the above step 1-2) is 2-20M.

[0023] Optionally, the molecular weight cut-off of the dialysis membrane used in dialysis in step 2) above is 8,000-20,000, preferably 9,000-18,000, preferably 10,000-17,000, preferably 11,000-16,000, preferably 12,000-15,000, for example, about 14,000.

[0024] The above injectable silk fibroin piezoelectric hydrogel can be prepared by a method comprising the following steps:

[0025] 1) Degum and dry natural mulberry cocoons to make degummed silk, dissolve the degummed silk, and prepare a stock solution of regenerated silk fibroin (RSF).

[0026] 2) Mix the stock solution of regenerated silk fibroin (RSF) obtained in step 1) with glycidyl methacrylate (GMA) and react to obtain an SF-MA solution, which is dialyzed and freeze-dried to obtain SF-MA powder.

[0027] 3) Dissolve the SF-MA powder in step 2) in water, add triethanolamine (TEA), N-vinyl-2-pyrrolidone (NVP), PVDF (polyvinylidene difluoride) spinning chips, and a photo-crosslinking agent L2959 (photoinitiator 2959, 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone), and crosslink by ultraviolet irradiation to obtain the injectable silk fibroin piezoelectric hydrogel.

[0028] In a specific embodiment, when the SF-MA powder in step 3) above is used, it is dissolved in water to prepare a solution with a concentration of 1-10 wt%, preferably 2-8 wt%, preferably 3-7 wt%, preferably 4-6 wt%, more preferably about 5 wt%.

[0029] The concentration of TEA is 10-100 mM, preferably 20-80 mM, preferably 25-70 mM, preferably 30-60 mM, more preferably about 40 mM.

[0030] The concentration of NVP is 30-300 mM, preferably 40-280 mM, preferably 50-250 mM, preferably 60-220 mM, preferably 70-200 mM, preferably 80-180 mM, more preferably about 100 mM.

[0031] The addition amount of PVDF spinning chips is 1-10 mg / ml, preferably 2-8 mg / ml, preferably 3-7 mg / ml, preferably 4-6 mg / ml, about 5 mg / ml.

[0032] The addition amount of the photo-crosslinking agent L2959 is 1-10 mg / ml, preferably 2-8 mg / ml, preferably 3-7 mg / ml, preferably 4-6 mg / ml, about 5 mg / ml.

[0033] It should be understood that when expressing numerical characteristics in this text, the term "about" or "around" means that the indicated number can have an error range or floating range of ±10%, ±9%, ±8%, ±7%, ±6% or ±5%.

[0034] Optionally, the preparation method of the PVDF spinning slice used in the above step 3) includes the following steps: dissolving PVDF in a mixed solvent with a volume ratio of DMSO (dimethyl sulfoxide) / AC (acetone) of about 7:3 to prepare a PVDF solution with a mass ratio of about 13 wt%, performing electrospinning under the conditions of a voltage of about 25 KV and a rate of about 1 ml / h to obtain a PVDF spinning membrane, and performing cryosectioning with a cryostat to prepare a PVDF spinning slice with a thickness of about 25 mm.

[0035] Another aspect of the present invention provides the use of the injectable silk fibroin piezoelectric hydrogel as described above in bone repair.

[0036] In a bone repair operation, after the bone injury site is exposed to the surgical field, the hydrogel liquid containing SF-MA, triethanolamine, N-vinyl-2-pyrrolidone, PVDF and the photo-crosslinking agent L2959 is injected into the bone injury site, and then irradiated with ultraviolet light for about 30-60 seconds, such as 40 seconds, and the hydrogel liquid can be shaped; then the wound can be sutured to quickly end the operation.

[0037] The present invention prepares a biodegradable and body-absorbable piezoelectric hydrogel based on silk protein. The silk fibroin piezoelectric hydrogel is colorless and transparent, can be injected into the bone defect site for bone repair treatment, and is shaped immediately during the operation, avoiding the estimation error of the bone injury site, shape and three-dimensional size that needs to be determined by imaging scanning before the surgical operation, greatly shortening the long cycle of preparing the colloidal mold, and improving the success rate and efficiency of the bone repair operation. The obtained piezoelectric hydrogel can generate microcurrents in response to self-generated movements or external stimuli (such as ultrasonic waves), regulate the physiological electrical microenvironment, thereby promoting bone regeneration, and the piezoelectric hydrogel has the efficacy of promoting angiogenesis, thereby promoting wound healing, and has the efficacy of immune regulation, thereby inhibiting the pro-inflammatory reaction and maintaining the homeostasis of the immune microenvironment. At the same time, it also has the efficacy of promoting cell antioxidant, can accelerate the postoperative recovery of patients, and has good clinical application prospects. Description of the Drawings

[0038] Figure 1 It is a physical photo of the shaped injectable silk fibroin piezoelectric hydrogel prepared by the present invention.

[0039] Figure 2 Shows the scanning electron microscope (SEM) photograph of the microstructure of the PVDF spun fibers prepared in Example 1.

[0040] Figure 3 Shows the scanning electron microscope (SEM) photograph of the microstructure of the injectable silk fibroin piezoelectric hydrogel.

[0041] Figure 4 Shows the test results of the piezoelectric properties of the injectable silk fibroin piezoelectric hydrogel with three PVDF spun fiber slice contents.

[0042] Figure 5 Shows the biocompatibility test results of the injectable silk fibroin piezoelectric hydrogel of the present invention. Fluorescence micrographs of cells after Calcein / PI staining of BMSCs cells co-cultured with different hydrogels and treatments for 1 d, 4 d, and 7 d.

[0043] Figure 6 Are the investigation photographs of the osteogenic efficacy of the injectable silk fibroin piezoelectric hydrogel prepared by the present invention. Among them, A are the macroscopic and microscopic pictures of ALP staining after osteogenic induction of BMSC cells (Bone Marrow-Derived Mesenchymal Stem Cells) in each group for 7 d; B are the macroscopic and microscopic pictures of ARS staining after osteogenic induction of BMSC cells in each group for 14 d; C is the expression level of intracellular osteogenic genes of BMSCs in each group after osteogenic induction for 7 d; D is the expression level of intracellular osteogenic proteins of BMSCs in each group after osteogenic induction for 7 d.

[0044] Figure 7 Are the investigation photographs of the angiogenesis efficacy of the silk fibroin PVDF piezoelectric hydrogel of the present invention. Among them, A are the micrographs of each group of HUVECS (Human umbilical vein endothelial cells) in the wound healing assay experiment; B are the micrographs of each group of HUVECS in the tube formation assay experiment.

[0045] Figure 8 Are the investigation photographs of the immunomodulatory efficacy of the silk fibroin PVDF piezoelectric hydrogel of the present invention. The pictures are the expression levels of pro-inflammatory genes and anti-inflammatory genes in RAW264.7 cells (mouse monocyte macrophage leukemia cells) after co-culture with different conditioned media.

[0046] Figure 9 Are the investigation photographs of the pro-cell antioxidant efficacy of the silk fibroin PVDF piezoelectric hydrogel of the present invention. The expression level of intracellular antioxidant proteins of BMSCs after co-culture for 7 d under different conditions. Detailed implementation mode

[0047] Silk fibroin (SF) has excellent toughness, biocompatibility, biodegradability, and thermal stability, so it has been widely studied and applied in the field of regenerative medicine. Piezoelectric materials can generate microcurrents in response to self-generated motion or external stimuli (such as ultrasonic waves) due to their unique properties. Piezoelectric materials are used at bone defect sites to regulate the physiological electrical microenvironment, thereby promoting bone regeneration.

[0048] Ordinary silk fibroin can accelerate the folding of β-sheets to form a gel by adjusting the environmental pH value, solution concentration, environmental temperature, etc., but this process usually takes more than 3 days. To accelerate the gelation rate, a tyrosinase and hydrogen peroxide system is often used to achieve chemical gelation, but it also takes 15 minutes. These existing silk fibroin hydrogel piezoelectric materials are obviously not ready-to-use when used in bone repair surgeries.

[0049] To enable the silk fibroin hydrogel piezoelectric material to gel faster during bone repair surgeries, reducing the exposure time of the surgical body part to air and the resulting damage. We use an ultraviolet light curing system for the colloidal setting of the silk fibroin hydrogel liquid. Through the L2959 photoinitiator, the silk fibroin hydrogel liquid can gel in about 40 seconds, greatly shortening the gelation time. At the same time, piezoelectric properties are imparted to the material by adding PVDF spinning. By itself, it can respond to mechanical activities to generate charges, promoting the proliferation and differentiation of stem cells and accelerating the repair of bone injuries.

[0050] The PVDF molecule is composed of C-F bonds and C-H bonds. The C-F bond is very polar, so PVDF has a high molecular polarity. In terms of crystal structure, PVDF can exist in three crystal phases: α-phase, β-phase, and γ-phase. Among them, the β-phase is the crystal structure with piezoelectric properties. Through the spinning process treatment of PVDF, the proportion of the β-phase is significantly increased, enhancing the piezoelectric properties.

[0051] The injectable silk fibroin piezoelectric hydrogel formed by the dispersion of PVDF spinning molecules in the silk protein hydrogel is a medical polymer composite material that can be slowly degraded and absorbed by the human body and is suitable for in vivo bone defect repair.

[0052] For the sake of convenient description, in this article, sometimes the "injectable silk fibroin piezoelectric hydrogel" is simply referred to as "silk protein PVDF piezoelectric hydrogel", "silk protein PVDF composite hydrogel", "composite piezoelectric hydrogel", and they have the same meaning.

[0053] Experimental results have shown that, in addition to promoting bone regeneration, the piezoelectric hydrogel of the present invention also has good angiogenesis efficacy, which further promotes wound healing. It also has immunomodulatory efficacy to inhibit pro-inflammatory responses and maintain the homeostasis of the immune microenvironment. Meanwhile, it has pro-cell antioxidant efficacy, which is beneficial to accelerating the postoperative recovery of patients.

[0054] The following further describes the present invention with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0055] Embodiment

[0056] In the embodiments of the present invention, if no specific description is made for the experimental operation temperature, the temperature generally refers to room temperature.

[0057] (15 - 35 °C).

[0058] In this article, the addition amounts, contents, and concentrations of various substances are involved. Among them, unless otherwise specified, the percentage content refers to the weight percentage content.

[0059] Unless otherwise specified, the "solution" mentioned in this article refers to an aqueous solution.

[0060] The chemical reagents used in the embodiments were purchased from Shanghai Chemical Reagent Company of China National Pharmaceutical Corporation and Shanghai Aladdin Biochemical Technology Co., Ltd.

[0061] PVDF, Arkema France, KR761.

[0062] Natural mulberry cocoons, Tongxiang Zhouquan Sangyi Silk Factory.

[0063] Embodiment 1: Preparation of regenerated silk fibroin stock solution

[0064] The preparation of the regenerated silk fibroin stock solution, which is the main component of the injectable silk fibroin piezoelectric hydrogel, includes the following steps:

[0065] Take anhydrous Na 2 CO 3 with the same mass as the silk, dissolve it in water, boil it at 100 °C for 30 min, and repeat twice.

[0066] Then wash it with distilled water and dry it in an oven at 37 °C for 3 days.

[0067] Dissolve the dried silk fibroin in a LiBr solution with a concentration of 9.3 mol / L at 60 °C and keep it warm for 1 hour.

[0068] Subsequently, 10 ml of GMA with a concentration of 705 mM was added, and the reaction was stirred at a rate of 400 rpm for 3 h.

[0069] The solution after the reaction was dialyzed for 3 days using a dialysis bag with a molecular weight cut-off of 140,000, concentrated by a PEG solution with a molecular weight of 20,000, and then freeze-dried to obtain SF-MA freeze-dried powder.

[0070] Example 2: Preparation of PVDF spinning slices

[0071] PVDF was dissolved in a solution with a DMSO / AC concentration ratio of 7:3 to prepare a PVDF solution with a mass ratio of 13 wt%. Electrospinning was carried out under the conditions of a voltage of 25 KV and a rate of 1 ml / h to obtain a PVDF electrospun membrane. PVDF spinning slices with a thickness of 25 mm were prepared by cryosectioning. The PVDF spinning slices were detected using a Hitachi S-4800 system, and the microstructural photographs are as Figure 2 shown. The photographs show the network structure of PVDF electrospinning.

[0072] Example 3: Preparation of silk fibroin PVDF piezoelectric hydrogel

[0073] 1. The SF-MA freeze-dried powder was dissolved in ultrapure water to prepare a solution with a concentration of 5 wt%. 40 mM TEA, 100 mM NVP, 5 mg / ml PVDF spinning slices, and 5 mg / ml photoinitiator L2959 were added and mixed evenly to obtain a silk fibroin piezoelectric hydrogel liquid. The liquid was poured into a small glass and cured by irradiating with ultraviolet light for 60 s to obtain an injectable silk fibroin piezoelectric hydrogel. The obtained hydrogel was a colorless and transparent colloid, as shown in Figure 1 . The content of PVDF in this hydrogel was 5 mg / ml, and it was named 5Mg-PVDF.

[0074] 2. Hydrogels were prepared in the same manner as in step 1, except that the addition amounts of PVDF spinning slices were adjusted to 10 mg / ml and 15 mg / ml respectively, and the corresponding colloids were named 10Mg-PVDF and 15Mg-PVDF respectively.

[0075] Scanning electron microscope photographs of the microstructure of the hydrogel without adding PVDF (SF-MA) and hydrogels containing different contents of PVDF (5Mg-PVDF, 10Mg-PVDF, and 15Mg-PVDF) are as Figure 3 shown. The photographs show the network structure of the cross-linking of silk fibroin fibers and PVDF electrospinning.

[0076] Example 4: Piezoelectric property test of silk fibroin PVDF piezoelectric hydrogel

[0077] The piezoelectric properties of the injectable silk fibroin piezoelectric hydrogels with different PVDF contents prepared in Example 3 were tested using a Keithley DMM6500 digital multimeter and an ultrasonic therapeutic apparatus made by Jili. The results are shown in Figure 4 .

[0078] The mechanical-electrical response signal was measured by a linkage device of an ultrasonic therapeutic apparatus and a high-precision general Keithley DMM6500 digital multimeter. The hydrogels with different PVDF contents could generate voltage under pressure, and as the PVDF content increased, the voltage of the hydrogel also increased accordingly. The hydrogels 5Mg-PVDF, 10Mg-PVDF, and 15Mg-PVDF generated voltages above 0.03V, 0.04V, and about 0.05V respectively.

[0079] Example 5: Investigation of the biocompatibility of silk fibroin PVDF piezoelectric hydrogels

[0080] The biocompatibility of the silk fibroin hydrogel piezoelectric materials with different PVDF contents was detected by the live / dead cell staining method.

[0081] The growth status of cells in each group was detected by the live / dead cell staining method: The number of BMSCs cells was adjusted to 1×10 5 cells / well and seeded in a 12-well plate. Live / dead cell staining was performed according to the instructions of the detection kit (Calcein / PI Cell Viability and Cytotoxicity Detection Kit, Beyotime, C2015S). The groups were: Ctrl (15Mg-PVDF hydrogel); ultrasound (15Mg-PVDF hydrogel + ultrasound); immersed medium (extract of 15Mg-PVDF hydrogel method); U+I (ultrasonic stimulation with the extract of 15Mg-PVDF hydrogel). After culturing for 1d, 4d, and 7d respectively, Calcein / PI staining was performed, and cell viability was observed under an inverted fluorescence microscope. The results are shown in Figure 5 , and the BMSCs cells in each culture plate grew well, indicating that the injectable silk fibroin PVDF piezoelectric hydrogel had no obvious toxicity and good biocompatibility.

[0082] Example 6: Investigation of the osteogenic efficacy of silk fibroin PVDF piezoelectric hydrogels

[0083] The osteogenic efficacy of the composite piezoelectric hydrogel was investigated by ALP staining method, ARS staining method, osteogenic gene qRT-PCR, and osteogenic protein immunoblotting experiment.

[0084] The osteogenic efficacy of the silk fibroin PVDF piezoelectric hydrogel was investigated by the ALP staining method: The BMSCs cells were adjusted to 1×10 5Cells were seeded at a density of -7 per well in 6-well plates and divided into the following groups: Ctrl group (without hydrogel), SF-MA group (hydrogel without PVDF), SF-MA+US group (SF-MA hydrogel treated with ultrasound), 15Mg-PVDF group (15Mg-PVDF hydrogel), and 15Mg-PVDF+US group (15Mg-PVDF hydrogel treated with ultrasound). Pre-prepared 20x osteogenic induction medium (α-MEM containing 10 Figure 6 mol / L dexamethasone, 10 mmol / L β-glycerophosphate, 50 μmol / L vitamin C, and 10% fetal bovine serum) was added to the culture medium of each group. The medium was changed every 2 days. After 7 days of osteogenic induction, ALP staining was performed according to the instructions of the detection kit (BCIP / NBT alkaline phosphatase color development kit, Beyotime, C3206). The results are shown in

[0085] Figure A. It was clearly visible both macroscopically and microscopically that the ALP staining in the 15Mg-PVDF+US group was deeper and the alkaline phosphatase activity was higher, indicating that the silk fibroin PVDF piezoelectric hydrogel after ultrasonic stimulation had a significant promoting effect on the osteogenic differentiation of BMSCs. 5 Cells were seeded at a density of -7 per well in 6-well plates and divided into the same groups as above. Pre-prepared 20x osteogenic induction medium (α-MEM containing 10 Figure 6 mol / L dexamethasone, 10 mmol / L β-glycerophosphate, 50 μmol / L vitamin C, and 10% fetal bovine serum) was added to the culture medium of each group. The medium was changed every 2 days. After 14 days of osteogenic induction, ARS staining was performed according to the instructions of the detection kit (Alizarin Red S staining solution (2%, pH 4.2; Beyotime)). The results are shown in

[0086] Figure B. It was clearly visible both macroscopically and microscopically that the ARS staining in the 15Mg-PVDF+US group was deeper, indicating that the silk fibroin PVDF piezoelectric hydrogel after ultrasonic stimulation had a significant promoting effect on the osteogenic differentiation of BMSCs. 5 Cells were seeded at a density of Figure 6 per well in 6-well plates and divided into the same groups as above. After co-culturing for 7 days, real-time reverse transcription PCR analysis was performed to detect the expression levels of osteogenic-related genes (Col1a1, OPN, Runx2, and β-actin as references). The results are shown in

[0087] Osteogenic protein immunoblotting experiment was used to investigate the osteogenic efficacy of silk protein PVDF piezoelectric hydrogel: The BMSCs cells were adjusted to 1×10 5 cells / well and seeded in 6-well plates. The grouping was the same as before. After co-culturing for 7 days, the cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing a protease and phosphatase inhibitor mixture, and the cellular proteins were extracted and quantified by Western blot analysis. Using the endogenous reference gene β-actin as a reference, the expressions of osteogenesis-related proteins OPN, Runx2, and Col1a1 were measured. The results are as Figure 6 shown in D below. It can be seen that the osteogenic protein expression level in the 15Mg-PVDF+US group was the highest, indicating that the silk protein PVDF piezoelectric hydrogel after ultrasonic stimulation had an obvious promoting effect on the osteogenic protein expression of BMSCs.

[0088] The results of ALP staining, ARS staining, osteogenic gene qRT-PCR, and osteogenic protein immunoblotting experiments all showed that the silk protein PVDF piezoelectric hydrogel had good osteogenic efficacy.

[0089] Example 7: Investigation of the angiogenesis efficacy of silk protein PVDF piezoelectric hydrogel

[0090] The wound healing assay and tube formation assay were used to evaluate the angiogenesis efficacy of silk protein PVDF piezoelectric hydrogel.

[0091] Wound healing assay: HUVECs were seeded in 24-well plates with 1×10 4 cells per well and incubated in an incubator at 37°C under 5% CO 2 atmosphere. The grouping was the same as before (Ctrl group (without hydrogel), SF-MA group (hydrogel without adding PVDF), SF-MA+US group (SF-MA treated with ultrasound), 15Mg-PVDF group (15Mg-PVDF hydrogel), 15Mg-PVDF+US group (15Mg-PVDF hydrogel treated with ultrasound)). After the cells were completely spread, a scratch was made directly on the cell layer using a 200 μl pipette tip. The suspended cells were washed with PBS. At 0 and 12 hours, the cells were observed under a microscope. The migration area was calculated using ImageJ. As Figure 7 shown in A below, it can be seen that the wound closure degree in the 15Mg-PVDF+US group was the highest, indicating that the silk protein PVDF piezoelectric hydrogel after ultrasonic stimulation significantly enhanced the angiogenesis of HUVECs in vitro.

[0092] Tube formation assay: 10 μL of Matrigel (Corning) was added to 6-well plates and allowed to solidify at 37°C for 30 minutes. Then, 2×10 4HUVECs were seeded in 6-well plates and grouped as before. After incubation for 12 hours, the tube formation of HUVECs was observed and imaged using a microscope. The number of tubular structures formed by HUVECs was blindly counted in randomly selected 4× microscope images of each group. The tube formation parameters were analyzed using ImageJ software. As Figure 7 shown in B, it can be seen that the connection and tube length in the 15Mg-PVDF+US group were significantly increased, indicating that the silk fibroin PVDF piezoelectric hydrogel after ultrasonic stimulation significantly enhanced the angiogenesis of HUVECs in vitro.

[0093] The results of the wound healing assay and tube formation experiment demonstrated that the silk fibroin PVDF piezoelectric hydrogel had good angiogenesis efficacy.

[0094] Example 8: Investigation of the immunomodulatory efficacy of silk fibroin PVDF piezoelectric hydrogel

[0095] The investigation of the immunomodulatory efficacy of silk fibroin PVDF piezoelectric hydrogel includes the following steps:

[0096] 1. Preparation of conditioned medium: The BMSCs were adjusted to 1×10 5 cells / well and seeded in 6-well plates, grouped as before. After co-culture for 3 days, the supernatant was obtained by centrifugation and filtration. The conditioned medium was composed of fresh medium mixed with the supernatant at a ratio of 1:1 (v / v).

[0097] 2. Intervention of conditioned medium on RAW264.7 cells: The RAW264.7 cells were adjusted to 1×10 5 cells / well and seeded in 6-well plates, and incubated at 37 °C under 5% CO 2 . Grouped as before.

[0098] 3. qRT-PCR was used to detect the expression of inflammation-related genes: Using β-actin as the endogenous reference gene, the expression levels of pro-inflammatory genes (CD86, iNOS) and anti-inflammatory genes (CD206, ARG1) were detected.

[0099] The results were as Figure 8 shown. The expression of pro-inflammatory genes (CD86, iNOS) decreased, and the expression of anti-inflammatory genes (CD206, ARG1) increased. Among them, the 15Mg-PVDF+US group showed the best performance, indicating that the silk fibroin PVDF piezoelectric hydrogel after ultrasonic stimulation could enhance the paracrine of BMSCs for immunomodulation, effectively regulating the polarization of M2 macrophages while reducing the M1 phenotype, thereby inhibiting the pro-inflammatory response and maintaining the homeostasis of the immune microenvironment.

[0100] Example 9: Investigation of the antioxidant efficacy of silk fibroin PVDF piezoelectric hydrogel

[0101] The antioxidant protein immunoblotting experiment was used to detect the promoting cell antioxidant efficacy of silk protein PVDF piezoelectric hydrogel.

[0102] The promoting cell antioxidant efficacy of silk protein PVDF piezoelectric hydrogel was investigated by the antioxidant protein immunoblotting experiment of BMSC cells: The BMSCs cells were adjusted to 1×10 5 cells / well and seeded in 6-well plates. The groups were: Ctrl (SF-MA), 10Mg-PVDF (10Mg-PVDF hydrogel), 10Mg-PVDF+U (10Mg-PVDF hydrogel was ultrasonically treated), luteolin (a kind of antioxidant compound). After co-culturing for 7 d, the cells were lysed with RIPA buffer (Beyotime, Shanghai, China) containing a protease and phosphatase inhibitor mixture, and the cell proteins were extracted and quantified by Western blot analysis. With the endogenous reference gene β-actin as a reference, the expressions of antioxidant-related proteins NRF2, NQO1, and GPX4 were measured. The results were as Figure 9 shown. It could be seen that the expression of antioxidant proteins in BMSC cells in the 10Mg-PVDF+U group increased, indicating that the silk protein PVDF piezoelectric hydrogel after ultrasonic stimulation could improve the antioxidant ability of BMSC cells, and the silk protein PVDF piezoelectric hydrogel had good antioxidant efficacy.

[0103] The above experimental results show that the silk protein PVDF piezoelectric hydrogel of the present invention has excellent piezoelectric effect, good biocompatibility, does not affect the growth of BMSCs cells, can efficiently promote the osteogenic differentiation, immunomodulation and improve the cell antioxidant ability of BMSCs under ultrasonic stimulation, can promote wound healing, and shows good clinical application prospects.

Claims

1. An injectable silk fibroin piezoelectric hydrogel, characterized in that: The invention comprises PVDF spinning and regenerated silk protein (SF-MA) with methacrylic anhydride groups, and the two are cross-linked to form a network structure.

2. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: The content of PVDF in the hydrogel is 5-15 mg / ml.

3. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: The regenerated silk protein with methacrylic anhydride groups is prepared by mixing a regenerated silk protein stock solution with glycidyl methacrylate and reacting them.

4. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: The raw materials of the injectable silk fibroin piezoelectric hydrogel also include triethanolamine, N-vinyl-2-pyrrolidone and photocrosslinking agent L2959.

5. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: The piezoelectric performance is: the mechanical-electrical response signal is measured by a mechanical testing machine and a KEITHLEY DMM6500 linkage device, and a voltage of 0.03V or more, preferably 0.04V or more is generated.

6. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: The regenerated silk protein with methacrylic anhydride groups (SF-MA) is prepared by a method comprising the following steps: 1) degumming and drying natural mulberry cocoons to obtain degummed silk, and dissolving the degummed silk to prepare a regenerated silk protein stock solution; 2) The prepared regenerated silk protein stock solution obtained in step 1) is mixed with glycidyl methacrylate and reacted to obtain SF-MA solution, which is dialyzed and freeze-dried to obtain SF-MA powder.

7. The injectable silk fibroin piezoelectric hydrogel according to claim 1, characterized in that: Prepared by a method comprising the following steps: 1) degumming and drying natural mulberry cocoons to obtain degummed silk, and dissolving the degummed silk to prepare a regenerated silk protein stock solution; 2) mixing the prepared regenerated silk protein stock solution obtained in step 1) with glycidyl methacrylate to react to obtain SF-MA solution, and dialyzing and freeze-drying to obtain SF-MA powder; 3) dissolving the SF-MA powder in step 2) in water, adding triethanolamine, N-vinyl-2-pyrrolidone, PVDF spinning chips and photocrosslinker L2959, and crosslinking by ultraviolet light to obtain the injectable silk fibroin piezoelectric hydrogel.

8. The injectable silk fibroin piezoelectric hydrogel according to claim 7, characterized in that: In step 3), the SF-MA powder is dissolved in water to prepare a solution with a concentration of 1-10 wt%; The concentration of TEA is 10-100 mM; The concentration of NVP is 30-300 mM; The amount of PVDF spinning chips added is 1-10 mg / ml; The amount of photocrosslinker L2959 added is 1-10 mg / ml.

9. The injectable silk fibroin piezoelectric hydrogel according to claim 7, characterized in that: The preparation method of the PVDF spinning slice used in step 3) comprises the following steps: dissolving PVDF in a mixed solvent of DMSO / AC with a volume ratio of 7:3 to prepare a PVDF solution with a mass ratio of 13wt%, performing electrospinning at a voltage of 25KV and a rate of 1ml / h to obtain a PVDF spinning membrane, and performing cryosectioning with a cryostat to prepare PVDF spinning slices.

10. Use of the injectable silk fibroin piezoelectric hydrogel according to any one of claims 1 to 9 in bone repair.

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