Novel spider silk fibroin cartilage repair gel scaffold and preparation method thereof

Abstract

The invention belongs to the field of biomedical materials and tissue engineering, and particularly relates to a composite gel scaffold based on verruca vestita spider silk protein and a preparation method of the composite gel scaffold, and the composite gel scaffold is used for repairing meniscus cartilage injury. According to the scaffold, the mechanical performance of spider silk protein, the microenvironment simulation function of chondroitin sulfate and the induced differentiation effect of BMP-2 growth factors are integrated, and in-situ regeneration of cartilage tissue is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials and tissue engineering, and particularly relates to a composite gel scaffold based on the silk fibroin protein of Macrothele wuliangshanensis and a preparation method thereof, which is used for repairing meniscus cartilage injury. Background Art

[0002] Meniscus cartilage injury is a common clinical disease. Traditional treatment methods (such as surgical resection or synthetic material implantation) have problems such as insufficient mechanical properties, poor biocompatibility, and inability to promote tissue regeneration. Existing scaffold materials (such as collagen, PLGA) have certain degradability, but their mechanical strength is low (compressive strength < 0.5 MPa), and growth factors (such as BMP-2) are easily inactivated due to the burst release effect, resulting in limited repair effects.

[0003] Silk fibroin protein has become an ideal scaffold material due to its natural high toughness (the fracture strength can reach 1.5 GPa), degradability, and low immunogenicity. However, single silk fibroin protein lacks the activity of promoting cartilage differentiation and needs to act synergistically with bioactive components. Chondroitin sulfate is the main component of cartilage matrix and can provide cell adhesion sites; BMP-2 can induce the differentiation of mesenchymal stem cells into cartilage, but its controlled release technology is still a difficult point.

[0004] In the prior art, CN106474536A discloses a silk fibroin protein - collagen composite scaffold, but its mechanical properties do not meet the requirements of the meniscus; CN108635656A proposes PLGA microspheres loaded with BMP-2, but the burst release rate is as high as 60% (within 24 hours).

[0005] Therefore, there is an urgent need for an effective scaffold material for meniscus cartilage injury. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a novel silk fibroin protein cartilage repair gel scaffold, which can realize the in-situ regeneration of cartilage tissue by integrating the mechanical properties of silk fibroin protein, the microenvironment simulation function of chondroitin sulfate, and the induction and differentiation effect of BMP-2 growth factor.

[0007] The present invention provides a silk fibroin protein composite gel, and the preparation method of the silk fibroin protein composite gel is as follows:

[0008] Step 1: Preparation of silk fibroin protein solution: Take freeze-dried silk fibroin protein and dissolve it in PBS, and stir until completely dissolved.

[0009] Step 2: Preparation of hyaluronic acid solution: Take hyaluronic acid and dissolve it in PBS, and stir overnight.

[0010] Step 3, preparation of the mixed solution: Mix the silk fibroin solution in Step 1 and the hyaluronic acid solution in Step 2 at a volume ratio of 6-8:2-4, and add chondroitin sulfate to make the final concentration of chondroitin sulfate 2-4%;

[0011] Step 4, cross-linking reaction: Mix the PBS solution of EDC and the PBS solution of NHS with the same molar concentration in equal volumes, then add PBS to supplement. The resulting solution is mixed with the mixed solution in Step 3, and stirred at 3-5 °C and 100-500 rpm for 10-15 h to form a homogeneous gel. Inject the resulting mixed solution into a mold and let it stand at 25-40 °C for 1-3 h to form a gel;

[0012] Step 5, washing: Rinse with PBS to remove unreacted cross-linking agent;

[0013] Step 6, sterilization treatment: Sterilize by ultraviolet irradiation.

[0014] Preferably, the preparation method of the silk fibroin composite gel is as follows:

[0015] Step 1, preparation of the silk fibroin solution: Dissolve 4 g of freeze-dried silk fibroin in 100 mL of PBS and stir until completely dissolved,

[0016] Step 2, preparation of the hyaluronic acid solution: Dissolve 3 g of hyaluronic acid in 100 mL of PBS and stir overnight;

[0017] Step 3, preparation of the mixed solution: Mix the silk fibroin solution in Step 1 and the hyaluronic acid solution in Step 2 at a volume ratio of 7:3. Take 100 mL of the mixed solution and add 3 g of chondroitin sulfate to make the final concentration of chondroitin sulfate 3%;

[0018] Step 4, cross-linking reaction: Mix the PBS solution of 50 mM EDC and the PBS solution of 50 mM NHS at a volume ratio of 1:1. The volumes of the PBS solution of EDC and the PBS solution of NHS are 25 ml respectively, and then add 50 ml of PBS to make 100 ml of solution. Mix with 100 ml of the mixed solution in Step 3, stir at 4 °C and 200 rpm for 12 h to form a homogeneous gel. Inject the resulting mixed solution into a mold and let it stand at 37 °C for 1 h to form a gel;

[0019] Step 5, washing: Rinse 3 times with PBS to remove unreacted cross-linking agent;

[0020] Step 6, sterilization treatment: Irradiate with ultraviolet light for 30 min, and the irradiation wavelength is 254 nm.

[0021] In some embodiments, Step 3 further includes adding BMP-2; preferably, the concentration of BMP-2 in the mixed solution in Step 3 is 30-60 ng / mL, and more preferably, the concentration of BMP-2 in the mixed solution in Step 3 is 50 ng / mL.

[0022] Preferably, the preparation method of the spider silk fibroin composite gel is as follows:

[0023] Step 1: Preparation of spider silk fibroin solution: Dissolve 4 g of freeze-dried spider silk protein in 100 mL of PBS, and stir until completely dissolved.

[0024] Step 2: Preparation of hyaluronic acid solution: Dissolve 3 g of hyaluronic acid in 100 mL of PBS and stir overnight.

[0025] Step 3: Preparation of the mixed solution: Mix the spider silk protein solution in Step 1 and the hyaluronic acid solution in Step 2 at a volume ratio of 7:3. Take 100 mL of the mixed solution, add 3 g of chondroitin sulfate to make the final concentration of chondroitin sulfate 3%, and add 5 μg of BMP-2 to make the final concentration 50 ng / mL.

[0026] Step 4: Cross-linking reaction: Mix the PBS solution of 50 mM EDC and the PBS solution of 50 mM NHS at a volume ratio of 1:1. The volumes of EDC and NHS are 25 mL respectively, and then add 50 mL of PBS to make 100 mL of solution. Mix it with 100 mL of the mixed solution in Step 3, stir at 4°C and 200 rpm for 12 h to form a homogeneous gel. Inject the obtained mixed solution into a mold and let it stand at 37°C for 1 h to form a gel.

[0027] Step 5: Washing: Rinse with PBS three times to remove the unreacted cross-linking agent.

[0028] Step 6: Sterilization treatment: Irradiate with ultraviolet light for 30 min at a wavelength of 254 nm.

[0029] In some embodiments, the pH of PBS is selected from 7 to 7.5; preferably 7.4.

[0030] In some embodiments, the mass ratio of the freeze-dried spider silk protein to hyaluronic acid is 3 - 4:3 - 4.

[0031] In some embodiments, the mass ratio of the freeze-dried spider silk protein, hyaluronic acid and chondroitin sulfate is 3 - 4:3 - 4:3 - 4.

[0032] In some embodiments, the mass ratio of the freeze-dried spider silk protein, hyaluronic acid, chondroitin sulfate and BMP-2 is 3 - 4:3 - 4:3 - 4:0.000005 - 0.00001.

[0033] In some embodiments, the preparation method of the freeze-dried spider silk protein is as follows:

[0034] Step 1: Weigh an appropriate amount of the silk of Macrothele wuliangshanensis, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight; weigh the dried silk, cut it into pieces, and place it in a round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2, boil it for 1 hour under standard atmospheric pressure, and then wash it several times with deionized water; repeat this process three times and dry it overnight at 65 °C to obtain degummed silk fibers;

[0035] Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) in a mass-to-volume ratio of 1:1 - 2 (mg / mL). Place the round-bottom flask containing the silk fiber - HFIP blend in a thermostatic magnetic stirrer and carry out condensation reflux at 58 °C and 500 rpm for 6 h. Filter it through a 400-mesh stainless steel sieve to obtain a natural silk fibroin solution, and rotary evaporate to remove HFIP;

[0036] Step 3: Prepare an aqueous solution of papain, where the mass-to-volume ratio of papain to water is 1:1 (mg / mL), and the mass-to-volume ratio of the dosage of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add it to the natural silk fibroin solution prepared in Step 2 for further enzymatic digestion. Carry out a water bath at 60 - 70 °C, with a pH of 7, and heat and digest for 120 min. Then, prepare a phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during the ultrafiltration process; Dilute the digested solution with the buffer first, place it in a 10 kDa ultrafiltration membrane to effectively retain the silk fibroin and allow the papain to pass through; Place the ultrafiltration membrane containing the residual protein in a centrifuge tube and repeat the following operation 3 times: Add 1 - 2 ml of PBS, use an ultrasonic disruptor with a power of 20%, in pulse mode, process for 30 seconds, centrifuge at 8000 rpm for 2 minutes, collect the solution, combine the solutions collected 3 times, and dry to obtain silk fibroin.

[0037] Preferably, the water bath is at 65 °C.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] The present invention realizes the sustained release of BMP-2 (7-day cumulative release rate < 30%) through a silk fibroin - hyaluronic acid gel system combined with covalent crosslinking technology, and at the same time provides the mechanical properties of natural cartilage-like (compressive strength ≥ 1.2 MPa).

[0040] Specific implementation cases

[0041] The following examples can enable those skilled in the art in this specialty to understand the present invention more comprehensively, but do not limit the present invention within the scope of the described examples.

[0042] Example 1: Extraction Technology of Spider Silk Fibroin

[0043] Step 1: Weigh an appropriate amount of the silk of Macrothele gigas from Wuliang Mountain, wash away the excess impurities with water, dry the washed silk in an oven at 50 °C, and place it in a drying oven to cool to a constant weight; weigh the dried silk, cut it into pieces, and place it in a 1.0 L round-bottom flask. Prepare a degumming solution with silk: sodium carbonate: water in a mass ratio of 1:2:2, boil it for 1 hour under standard atmospheric pressure, and then wash it several times with deionized water; repeat this process three times, and dry it overnight at a temperature of 65 °C to obtain degummed silk fibers;

[0044] Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) in a mass-volume ratio of 1:1 - 2 (mg / mL). Place the round-bottom flask containing the silk fiber - HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58 °C and 500 rpm for 6 h. Filter it through a 400-mesh stainless steel sieve to obtain a natural spider silk protein solution, and rotary evaporate to remove HFIP;

[0045] Step 3: Prepare an aqueous solution of papain, where the mass-volume ratio of papain to water is 1:1 (mg / mL), and the mass-volume ratio of the dosage of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add it to the natural spider silk protein solution prepared with HFIP in advance for further enzymatic digestion. Carry out water bath at 65 °C, pH = 7, heat and enzymatically digest for 120 min. Then, prepare a phosphate buffer solution (PBS) with pH = 7.3 for sample dilution and membrane cleaning during the ultrafiltration process; dilute the enzymatically digested solution with the buffer first, put it into an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to effectively retain the spider silk fibroin and allow the papain to pass through; place the ultrafiltration membrane containing the residual protein in a centrifuge tube, and repeat the following operations 3 times: add 1 - 2 mL of PBS, use an ultrasonic disruptor with a power of 20%, pulse mode, process for 30 seconds, centrifuge at 8000 rpm for 2 minutes, collect the solution, combine the solutions collected 3 times, and dry to obtain fibroin.

[0046] Example 2: Spider Silk Fibroin Gel Group

[0047] 1. Solution Preparation

[0048] Spider silk protein solution: Weigh 4 g of freeze-dried spider silk protein, add 100 mL of phosphate buffer solution (PBS, pH 7.4), and stir magnetically until completely dissolved to prepare a 4% w / v solution.

[0049] Hyaluronic acid solution: Weigh 3 g of hyaluronic acid, add 100 mL of PBS (pH 7.4), stir overnight, and prepare a 3% w / v solution.

[0050] 2. Mixing

[0051] Mix in a volume ratio of 7:3 (spider silk protein solution: hyaluronic acid solution), and take 100 mL of the mixed solution.

[0052] 3. Cross-linking reaction

[0053] Add 25 mL each of 50 mM EDC in PBS solution and 50 mM NHS in PBS solution (volume ratio 1:1), then add 50 mL of PBS to make 100 mL of solution and add it to 100 mL of the mixed solution. Stir at 4 °C and 200 rpm for 12 hours to form a homogeneous gel. Inject the mixed solution into a cylindrical mold with a diameter of 5 mm and let it stand at 37 °C for 1 hour to form a gel.

[0054] 4. Post-treatment

[0055] Washing: Rinse the gel 3 times with PBS to remove unreacted cross-linking agent.

[0056] Sterilization: Irradiate with ultraviolet light (254 nm) for 30 minutes and store in PBS at 4 °C for later use.

[0057] Example 3: Spider silk fibroin gel + chondroitin sulfate gel group

[0058] 1. Solution preparation

[0059] Spider silk protein solution: 4% w / v (4 g of freeze-dried spider silk protein + 100 mL of PBS, pH 7.4), stir magnetically until completely dissolved.

[0060] Hyaluronic acid solution: 3% w / v (3 g of hyaluronic acid + 100 mL of PBS, pH 7.4), stir overnight.

[0061] 2. Mixing and adding chondroitin sulfate

[0062] Mix the spider silk protein solution and the hyaluronic acid solution in a volume ratio of 7:3, take 100 mL of the mixed solution, add 3 g of chondroitin sulfate powder, and stir until completely dissolved. The final concentration is 3% w / v.

[0063] 3. Cross-linking reaction

[0064] Add 25 mL each of 50 mM EDC in PBS solution and 50 mM NHS in PBS solution (volume ratio 1:1), then add 50 mL of PBS to make 100 mL of solution and add it to 100 mL of the mixed solution. Stir at 4 °C and 200 rpm for 12 hours to form a homogeneous gel. Inject the mixed solution into a mold (cylindrical with a diameter of 5 mm) and let it stand at 37 °C for 1 hour to form a gel.

[0065] 4. Post-treatment

[0066] Washing: Rinse 3 times with PBS to remove unreacted cross-linking agent.

[0067] Sterilization: Irradiate with ultraviolet light for 30 minutes (254 nm) and store in PBS at 4°C.

[0068] Example 4: Preparation of Composite Gel of Spider Silk Fibroin + Chondroitin Sulfate + BMP-2

[0069] 1. Solution Preparation:

[0070] Spider silk fibroin solution: 4% w / v: 4 g of freeze-dried spider silk protein + 100 mL of PBS, pH 7.4, stir magnetically until completely dissolved.

[0071] Hyaluronic acid solution: 3% w / v: 3 g of hyaluronic acid + 100 mL of PBS, pH 7.4, stir overnight.

[0072] 2. Mixing:

[0073] Mix according to the volume ratio of spider silk protein solution: hyaluronic acid solution = 7:3. Take 100 mL of the mixed solution, add 3 g of chondroitin sulfate to make the final concentration of chondroitin sulfate 3% and 5 μg of BMP-2 to make the final concentration 50 ng / mL.

[0074] 3. Crosslinking Reaction:

[0075] Add 25 mL each of 50 mM EDC in PBS solution and 50 mM NHS in PBS solution (volume ratio 1:1), then add 50 mL of PBS to make 100 mL of solution and add it to 100 mL of the mixed solution. Stir at 4°C and 200 rpm for 12 h to form a homogeneous gel. Inject the mixed solution into a mold (5 mm diameter cylinder) and let it stand at 37°C for 1 h to form a gel.

[0076] 4. Washing:

[0077] Rinse with PBS three times to remove unreacted crosslinking agent.

[0078] 5. Sterilization Treatment:

[0079] Irradiate with ultraviolet light for 30 min (254 nm) and store in PBS at 4°C.

[0080] Example 5: In Vitro Gel Performance Test of Examples 2 - 4

[0081] 5.1 Steps for Gel Performance Test (1) Compressive Strength Test

[0082] Standard: ASTM D695 (Test for Compressive Properties of Materials).

[0083] Instrument: Universal Material Testing Machine (Instron 5567).

[0084] Steps:

[0085] Cylindrical gel samples with a diameter of 5 mm and a height of 3 mm were prepared (n=5).

[0086] The compression rate was 1 mm / min, and the maximum compression force (F) and deformation (ΔL) were recorded.

[0087] Calculation formula:

[0088]

[0089] (2) Porosity measurement (ethanol replacement method)

[0090] step:

[0091] Weigh the dry gel mass (W dry W dry ).

[0092] Immerse in ethanol for 24 hours until saturated, and weigh the wet gel mass (W we tW wet ).

[0093] Calculation formula:

[0094]

[0095] (Pethanol=0.789g / ㎝3,V gel =πr 2 h)

[0096] (3) BMP-2 loading and release rate

[0097] Drug loading determination:

[0098] Standard curve: ELISA kit (Human BMP-2 Quantikine ELISA Kit) was used to draw a 0-100 ng / mL standard curve (R 2 >0.99).

[0099] Sample treatment: 1 g of gel was dissolved in 1 mL of collagenase (2 mg / mL, 37°C for 24 h), and the supernatant was collected by centrifugation.

[0100] calculate:

[0101]

[0102] Cumulative release rate:

[0103] The gel was immersed in 10 mL of PBS (pH 7.4) and incubated at 37°C in a shaker (50 rpm).

[0104] Samples were taken at regular intervals (1, 3, 5, and 7 days), and the release amount was detected by ELISA.

[0105] Calculation formula:

[0106]

[0107] (4) Detection of cell viability by CCK-8

[0108] Reagent: CCK-8 kit (Dojindo CK04).

[0109] Steps:

[0110] 1. Aspirate the culture medium, and add 10 μL of CCK-8 reagent + 90 μL of DMEM to each well.

[0111] 2. Incubate at 37 °C in the dark for 2 h.

[0112] 3. Measure the absorbance at 450 nm (OD value) with an enzyme-linked immunosorbent assay (ELISA) reader.

[0113] Calculation formula:

[0114]

[0115] Blank well: Only contains CCK-8 reagent + DMEM (without cells).

[0116] Control group: Normal BMSCs without adding the extract solution (100% viability).

[0117] (5) Detection of SOX9 and Aggrecan mRNA expression levels

[0118] 1. Cell culture and grouping treatment:

[0119] Take rabbit bone marrow mesenchymal stem cells and inoculate them in a 6-well plate (density 1×10 5 cells / well), use DMEM medium containing 10% fetal bovine serum, and culture at 37 °C and 5% CO2 until 80% confluence, then perform the following grouping treatment:

[0120] Blank control group: Replace with fresh medium;

[0121] Commercial collagen group: Add the collagen scaffold extract solution prepared according to ISO 10993-12 standard;

[0122] Experimental group: Add the composite gel extract solutions of Example 2 (spider silk protein), Example 3 (spider silk protein + chondroitin sulfate), and Example 4 (spider silk protein + chondroitin sulfate + BMP-2) respectively.

[0123] 2. RNA extraction and reverse transcription:

[0124] Extract total RNA and synthesize cDNA according to the following steps:

[0125] a) Lysis: After discarding the culture medium, wash the cells with PBS, add 1 mL of TRIzol reagent to each well and let it stand for 5 minutes;

[0126] b) Layering: Add 0.2 mL of chloroform, shake vigorously for 15 seconds, and centrifuge at 4°C, 12,000 rpm for 15 minutes;

[0127] c) Precipitation: Transfer the upper aqueous phase to a new tube, add 0.5 mL of isopropanol, let it stand for 10 minutes, and centrifuge at 4°C, 12,000 rpm for 10 minutes;

[0128] d) Purification: Wash the precipitate with 75% ethanol, dissolve it in 20 μL of DEPC water after centrifugation, and measure the RNA concentration with NanoDrop (A260 / A280 = 1.8 - 2.0);

[0129] e) Reverse transcription: Take 1 μg of RNA and construct cDNA using M-MLV reverse transcriptase (reaction conditions: 42°C for 60 minutes → 70°C for 5 minutes).

[0130] 3. Real-time fluorescence quantitative PCR (qPCR)

[0131] Primer design: SOX9: Forward: 5'-AGGAAGCTCGCGGACCAGTAC-3'

[0132] Reverse: 5'-TGTGCAGGTGCGGGTACTGG-3'

[0133] Aggrecan: Forward: 5'-TGCGCTACCGAGACATCATTG-3'

[0134] Reverse: 5'-TCGTCCTCGTCCTCCAGTTT-3'

[0135] Internal reference gene (GAPDH): Forward: 5'-GAAGGTGAAGGTCGGAGTC-3'

[0136] Reverse: 5'-GAAGATGGTGATGGGATTTC-3'

[0137] Reaction system (20 μL): SYBR Green Master Mix: 10 μL cDNA template: 2 μL Forward and reverse primers (10 μM): 0.5 μL each DEPC water: 7 μL Reaction program: 95°C pre-denaturation: 5 minutes 95°C denaturation: 15 seconds 60°C annealing / extension: 30 seconds (40 cycles) Melting curve analysis: 65°C - 95°C, increasing temperature by 0.5°C every 5 seconds.

[0138] 4. Relative quantitative data analysis: The 2-ΔΔCt method was used to calculate gene expression levels. ΔCt = Ct(target gene) - Ct(GAPDH); ΔΔCt = ΔCt(treatment group) - ΔCt(blank control group); Relative expression level = 2-ΔΔCt. Statistical analysis: One-way ANOVA analysis was performed using SPSS 20.0. The data were expressed as mean ± standard deviation, and a significant difference was defined as P < 0.05.

[0139] The gene expression levels were calculated by the 2-ΔΔCt method, where:

[0140] ΔCt = Ct(target gene) - Ct(GAPDH)

[0141] ΔΔCt = ΔCt(treatment group) - ΔCt(blank control group)

[0142] Statistical analysis was performed using one-way ANOVA with SPSS 20.0, and the significance threshold was set at P < 0.05.

[0143] (6) Key data of gel properties and their significance

[0144]

[0145]

[0146] 5.2 Gel property test results

[0147] 5.2.1 Safety test

[0148] As shown in Table 1, in the cytotoxicity test (biocompatibility), the CCK-8 method showed that the survival rate of the spider silk protein gel of the present invention was > 95% at 7 days (ISO 10993-5), meeting the non-toxicity standard of ISO 10993-5.

[0149] Table 1 Cell safety performance test among different groups

[0150]

[0151] Note: Compared with the commercial collagen group, P* < 0.05, P** < 0.01.

[0152] The commercial collagen group was purchased from Beijing Qunxiao Keyuan Biotechnology Co., Ltd., and the collagen scaffold had a model number of GSB010620.

[0153] 5.2.2 SOX9 and Aggrecan mRNA expression levels

[0154] As shown in Table 2, the qPCR results showed that the expression levels of SOX9 and Aggrecan mRNA were increased by 3.2-fold compared with the commercial collagen scaffold group (P < 0.01).

[0155] Table 2 SOX9 and Aggrecan mRNA expression levels among different groups (n = 3)

[0156]

[0157]

[0158] Note: Compared with the commercial collagen group, P* < 0.05, P** < 0.01

[0159] 5.2.3 Gel compressive strength, porosity, BMP-2 sustained-release performance, and drug loading capacity prepared in Example 4

[0160] Compressive strength: 1.35 ± 0.12 MPa (ASTM D695 standard, n = 5).

[0161] Porosity: 82.5 ± 3.2% (ethanol displacement method).

[0162] BMP-2 sustained release: In vitro PBS release experiment: The cumulative release rate in 7 days was 28.4 ± 2.1% (ELISA detection, n = 3), significantly better than the prior art (such as the burst release rate of PLGA microspheres being more than 60%), indicating that the cross-linking system effectively controls drug release.

[0163] BMP-2 drug loading capacity data are as follows: As shown in Table 3, the drug loading capacity of the gel in Example 4 group was measured to be 4.8 ± 0.3 μg / g (i.e., each gram of gel contains 4.8 μg of BMP-2) after the gel was dissolved, meeting the design target (5 μg / g).

[0164] Table 3 BMP-2 drug loading capacity and sustained-release performance among different groups (n = 3)

[0165]

[0166] The above test results of the present invention were analyzed using SPSS 20.0 statistical software. One-way ANOVA was used for comparison among multiple groups, and the data results were expressed as ; P < 0.05 indicates that the difference is statistically significant.

[0167] In vivo performance test of different gel groups in Example 6

[0168] 6.1 Modeling steps

[0169] Experimental animal preparation: Healthy adult New Zealand white rabbits (weight 2.5 - 3.0 kg, n = 24) were selected, adaptively fed for 1 week, and allowed free access to food and water. They were fasted for 12 hours before the operation and allowed free access to water.

[0170] Anesthesia and disinfection: Anesthesia was induced by intramuscular injection of Zoletil (0.1 mL / kg) combined with ketamine (20 mg / kg).

[0171] The right knee was shaved, disinfected with povidone-iodine, and covered with a sterile drape.

[0172] Establishment of the meniscus defect model: A lateral knee incision (about 2 cm) was made to expose the meniscus. A full-thickness defect (3 mm in depth) was created in the avascular area of the meniscus using a 3-mm diameter circular cutter. The surgical field was rinsed with normal saline to remove debris.

[0173] 6.2 Grouping: Blank control group, commercial collagen scaffold group (Beijing Qunxiao Keyuan Biotechnology Co., Ltd., collagen scaffold, model number GSB010620), silk gel group, silk gel + chondroitin sulfate CS group, silk gel + chondroitin sulfate CS + BMP-2 group (n = 6).

[0174] 6.3 Administration steps

[0175] Gel implantation method:

[0176] Blank control group: Only the defect was created, and no material was implanted.

[0177] Commercial collagen group: The commercial collagen gel (3 mm in diameter, 3 mm in height) was filled into the defect.

[0178] Example group (silk protein composite gel):

[0179] Example 2 (silk gel group): Liquid silk protein gel (4% w / v) was directly injected and allowed to gel at 37°C for 1 hour.

[0180] Example 3 (silk + chondroitin sulfate group) and Example 4 (silk + chondroitin sulfate + BMP-2 group): The preformed cylindrical gel (3 mm in diameter, 3 mm in height) was implanted into the defect.

[0181] The gel was gently pressed to make it fit with the surrounding tissues to avoid displacement.

[0182] Postoperative treatment: The joint capsule and skin were sutured layer by layer. Penicillin (50,000 units / kg) was intramuscularly injected for 3 consecutive days after surgery to prevent infection.

[0183] The animals were allowed free movement, and the wound healing was observed regularly.

[0184] 6.4 Detection steps

[0185] Postoperative observation and sample collection: Time points: 4, 8, and 12 weeks after surgery. Six animals in each group were sacrificed (n = 6 / group).

[0186] Specimen collection: The right knee meniscus and surrounding tissues were taken, fixed in 4% paraformaldehyde for 24 hours, dehydrated, and embedded in paraffin.

[0187] Histological analysis: HE staining

[0188] The section thickness was 5 μm, and the tissue structure was observed by HE staining

[0189] Microscopic scoring (ICRS standard): surface flatness, cell distribution, matrix staining, cartilage thickness (total score 0 - 12 points).

[0190] ICRS scoring criteria

[0191] Scoring dimensions:

[0192] Surface flatness (0 - 3 points): 0 = defect, 3 = smooth.

[0193] Cell distribution (0 - 3 points): 0 = no cells, 3 = uniform chondrocytes.

[0194] Matrix staining (0 - 3 points): 0 = no type II collagen, 3 = strong Safranin O staining.

[0195] Cartilage thickness (0 - 3 points): 0 = no new tissue, 3 = consistent with the surrounding cartilage.

[0196] Total score: 0 - 12 points, ≥9 points is excellent repair.

[0197] Immunohistochemistry (type II collagen):

[0198] Type II collagen antibody (Abcam ab34712) was used, DAB color development, and the proportion of positive area was analyzed by ImageJ.

[0199] Imaging detection (MRI):

[0200] Parameters: 3.0T MRI (T2-weighted sequence, slice thickness 1 mm), measuring the thickness and signal intensity of the new cartilage.

[0201] Analysis: Cartilage thickness ≥2.0 mm is qualified for repair, and the signal uniformity is used to evaluate the matrix maturity.

[0202] Statistical processing: Data were expressed as mean ± standard deviation, analyzed by one-way ANOVA with SPSS 20.0, and the inter-group difference with P < 0.05 was considered significant.

[0203] Detection indexes:

[0204] Immunohistochemistry: Proportion of positive area of type II collagen (analyzed by ImageJ).

[0205] MRI detection: 3.0T MRI scan, measuring the thickness of the new cartilage.

[0206] Table 4: ICRS scores and cartilage thickness in different groups (12 weeks after surgery)

[0207]

[0208] Note: Compared with the commercial collagen group, P* < 0.05, P** < 0.01. Compared with the silk gel group of Example 2, P # <0.05, P ## <0.01

[0209] Through standardized modeling and detection processes, this experiment verified the in vivo repair efficacy of the silk protein composite gel in the Example 4 group. Its ICRS score (9.6 ± 1.1 points) and the thickness of newly formed cartilage (2.3 ± 0.6 mm) were significantly better than those of the commercial control group (P < 0.01), meeting the clinical repair requirements. The proportion of type II collagen positive area in the Example 4 group reached 75.3 ± 6.2%, significantly higher than that of the commercial group (32.1 ± 5.4%). Moreover, the Example 4 group and the Example 3 group were significantly better than the Example 2 group in terms of ICRS score, thickness of newly formed cartilage, and type II collagen positive area.

[0210] Example 7 Degradation ability and safety test of Example 4

[0211] 7.1 The following data are based on in vitro (simulated body fluid) and in vivo (animal model) degradation experiments to show the degradation performance of the silk protein composite gel scaffold.

[0212] 7.1.1 Test grouping:

[0213] The degradation experiment was divided into two groups: in vitro simulated degradation (PBS environment) and in vivo animal model degradation. The specific grouping is as follows:

[0214] In vitro experimental group (n = 5): Example 4 composite gel group (silk protein + chondroitin sulfate + BMP-2), commercial collagen group (control)

[0215] In vivo experimental group (n = 6): Example 4 composite gel group (silk protein + chondroitin sulfate + BMP-2), blank control group, commercial collagen group (control)

[0216] 7.1.2 Test steps:

[0217] Sample preparation:

[0218] Prepare cylindrical gel samples with a diameter of 5 mm and a height of 3 mm (in vitro experiment) or implant them into the meniscus defect of animals (in vivo experiment).

[0219] Degradation environment setting:

[0220] In vitro: Immerse the sample in 10 mL of PBS buffer (pH 7.4, 37 °C) and place it on a thermostatic shaker (50 rpm).

[0221] In vivo: Implant it surgically into the meniscus defect of New Zealand white rabbits and observe regularly after the operation.

[0222] Data collection:

[0223] Residual mass percentage: Regularly take out the samples (in vitro: 0, 4, 8, 12 weeks; in vivo: 4, 8, 12 weeks after the operation), weigh them after drying to a constant weight, and calculate the degradation rate.

[0224] Morphological change analysis: Use a scanning electron microscope (SEM) to observe the changes in the surface and pore structure of the samples (in vitro experiment).

[0225] Histological detection: In the in vivo experiment, evaluate the synchrony of scaffold degradation and cartilage regeneration through tissue sections (HE staining, Safranin O staining).

[0226] Table 5: In vitro degradation rate of Example 4 (PBS, 37 °C, n = 5)

[0227]

[0228] Table 6: In vitro degradation rate data of commercial collagen (PBS, 37 °C, n = 5) Time (weeks) Residual mass percentage (%) Degradation rate (%) Morphological change (observed by SEM)

[0229]

[0230] The experiment verified the matching of the degradation rate of the composite gel with cartilage regeneration by comparing Example 4 with the commercial group (in vitro 12-week degradation rate of 62.3%). By regulating the cross-linking density and composition ratio (such as silk fibroin:chondroitin sulfate = 7:3), the composite gel made the degradation curve coincide with the cartilage regeneration process, solving the problem of "too fast or too slow degradation" of traditional materials and being an ideal scaffold for meniscus repair.

[0231] In the in vivo experiment, the scaffold in the Example 4 group was completely degraded 12 weeks after the operation, and the thickness of the newly formed cartilage reached 2.3 ± 0.6 mm (ICRS score of 9.6 points), which was significantly better than the commercial control group.

[0232] Table 7: Evaluation of in vivo degradation and cartilage regeneration effects (n = 5)

[0233]

[0234]

[0235] 7.2 In vivo safety test of the Example 4 group

[0236] There were no significant differences in the concentrations of IL-6 and TNF-α between the 4 groups of the examples and the commercial group (P = 0.32 and P = 0.45), indicating that the composite gel did not trigger additional inflammatory responses. The detection of inflammatory factors further supported the clinical applicability of the composite gel in Example 4. Its immune response was comparable to that of the commercial collagen scaffold, without potential toxicity risks, and its in vivo safety met the ISO 10993-6 biocompatibility standard.

[0237] Table 8: Local inflammatory factor levels at 12 weeks after surgery (n = 6)

[0238] Group IL-6 (pg / mg protein) TNF-α (pg / mg protein) Blank control group 12.3±2.1 8.5±1.7 Commercial collagen group 18.6±3.5 10.2±2.3 Example 4 spider silk + chondroitin sulfate + BMP-2 group 17.9±2.8 9.8±1.9

[0239] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A silk fibroin composite gel, and the preparation method of the silk fibroin composite gel is as follows: Step 1: Preparation of silk fibroin protein solution: Take freeze-dried silk protein and dissolve it in PBS, and stir until completely dissolved. Step 2: Preparation of hyaluronic acid solution: Take hyaluronic acid and dissolve it in PBS, and stir overnight. Step 3: Preparation of mixed solution: Mix the silk fibroin protein solution in Step 1 with the hyaluronic acid solution in Step 2 at a volume ratio of 6-8:2-4, and add chondroitin sulfate to make the final concentration of chondroitin sulfate 2-4%. Step 4: Crosslinking reaction: Mix the PBS solution of EDC with the same molar concentration and the PBS solution of NHS in equal volumes, and then add PBS to supplement. The obtained solution is mixed with the mixed solution in Step 3, and stirred at 3-5 °C and 100-500 rpm for 10-15 h to form a homogeneous gel. Inject the obtained mixed solution into a mold and let it stand at 25-40 °C for 1-3 h to form a gel. Step 5: Washing: Rinse with PBS to remove unreacted crosslinking agent. Step 6: Sterilization treatment: Sterilize by ultraviolet irradiation.

2. The silk fibroin composite gel according to claim 1, wherein Step 3 further comprises adding BMP-2; preferably, the concentration of BMP-2 in the mixed solution in Step 3 is 30-60 ng / mL, and more preferably, the concentration of BMP-2 in the mixed solution in Step 3 is 50 ng / mL.

3. The silk fibroin composite gel according to claim 1, and the preparation method of the silk fibroin composite gel is as follows: Step 1: Preparation of silk fibroin protein solution: Take 4 g of freeze-dried silk protein and dissolve it in 100 mL of PBS, and stir until completely dissolved. Step 2: Preparation of hyaluronic acid solution: Take 3 g of hyaluronic acid and dissolve it in 100 mL of PBS, and stir overnight. Step 3: Preparation of mixed solution: Mix the silk fibroin protein solution in Step 1 with the hyaluronic acid solution in Step 2 at a volume ratio of 7:

3. Take 100 mL of the mixed solution, and add 3 g of chondroitin sulfate to make the final concentration of chondroitin sulfate 3%. Step 4: Crosslinking reaction: Mix the PBS solution of 50 mM EDC and the PBS solution of 50 mM NHS at a volume ratio of 1:

1. The volumes of the PBS solution of EDC and the PBS solution of NHS are 25 mL respectively, and then add 50 mL of PBS to make 100 mL of solution. Mix it with 100 mL of the mixed solution in Step 3, stir at 4 °C and 200 rpm for 12 h to form a homogeneous gel. Inject the obtained mixed solution into a mold and let it stand at 37 °C for 1 h to form a gel. Step 5: Washing: Rinse 3 times with PBS to remove unreacted crosslinking agent. Step 6: Sterilization treatment: Irradiate with ultraviolet light for 30 min, and the irradiation wavelength is 254 nm.

4. The silk fibroin composite gel according to claim 2, and the preparation method of the silk fibroin composite gel is as follows: Step 1: Preparation of silk fibroin protein solution: Take 4 g of freeze-dried silk protein and dissolve it in 100 mL of PBS, and stir until completely dissolved. Step 2: Preparation of hyaluronic acid solution: Take 3 g of hyaluronic acid and dissolve it in 100 mL of PBS, and stir overnight. Step 3, Preparation of the mixed solution: Mix the silk fibroin solution in Step 1 and the hyaluronic acid solution in Step 2 at a volume ratio of 7:

3. Take 100 mL of the mixed solution, add 3 g of chondroitin sulfate to make the final concentration of chondroitin sulfate 3% and 5 μg of BMP-2 to make the final concentration 50 ng / mL; Step 4, Crosslinking reaction: Mix the PBS solution of 50 mM EDC and the PBS solution of 50 mM NHS at a volume ratio of 1:

1. The volumes of the EDC PBS solution and the NHS PBS solution are 25 ml respectively, and then add 50 ml of PBS to make 100 ml of solution. Mix it with 100 ml of the mixed solution in Step 3, stir at 4°C and 200 rpm for 12 h to form a homogeneous gel. Inject the obtained mixed solution into a mold and let it stand at 37°C for 1 h to form a gel; Step 5, Washing: Rinse with PBS three times to remove the unreacted crosslinking agent; Step 6, Sterilization treatment: Irradiate with ultraviolet light for 30 min, and the irradiation wavelength is 254 nm.

5. The silk fibroin protein composite gel according to any one of claims 1-4, wherein the pH of the PBS is selected from 7 to 7.5; preferably 7.

4.

6. The silk fibroin protein composite gel according to any one of claims 1-4, wherein the mass ratio of the freeze-dried silk fibroin to hyaluronic acid is 3-4:3-4.

7. The silk fibroin protein composite gel according to any one of claims 1-4, wherein the mass ratio of the freeze-dried silk fibroin, hyaluronic acid and chondroitin sulfate is 3-4:3-4:3-4.

8. The silk fibroin protein composite gel according to any one of claims 1-4, wherein the mass ratio of the freeze-dried silk fibroin, hyaluronic acid, chondroitin sulfate and BMP-2 is 3-4:3-4:3-4:0.000005-0.00001.

9. The silk fibroin protein composite gel according to any one of claims 1-4, wherein the preparation method of the freeze-dried silk fibroin is as follows: Step 1: Weigh an appropriate amount of the silk of Macrothele wuliangshanensis, wash away the excess impurities with water, dry the washed silk in an oven at 50°C, and place it in a drying oven to cool to a constant weight; Weigh the dried silk, cut it into pieces, and place it in a round-bottom flask. Make a degumming solution with silk: sodium carbonate: water at a mass ratio of 1:2:2, boil it under standard atmospheric pressure for 1 hour, and then wash it several times with deionized water; Repeat this process three times and dry it overnight at 65°C to obtain degummed silk fibers; Step 2: Add the degummed silk fibers and HFIP to the hexafluoroisopropanol solvent (HFIP) at a mass-volume ratio of 1:1-2 (mg / mL). Place the round-bottom flask containing the silk fiber-HFIP blend in a thermostatic magnetic stirrer, and carry out condensation reflux at 58°C and 500 rpm for 6 h. Filter it through a 400-mesh stainless steel sieve to obtain a natural silk fibroin solution, and rotary evaporate to remove HFIP; Step 3: Prepare an aqueous papain solution using papain, where the mass-volume ratio of papain to water is 1:1 (mg / mL), and the mass-volume ratio of papain to HFIP is 1:0.625 - 2.5 (mg / mL). Add the natural silk fibroin protein solution prepared in Step 2 for further enzymatic digestion. Carry out a water bath at 60 - 70 °C, with a pH of 7. After heating and enzymatic digestion for 120 min, prepare a phosphate buffer solution (PBS) with a pH of 7.3 for sample dilution and membrane cleaning during the ultrafiltration process. Dilute the enzymatically digested solution with the buffer first, and place it into an ultrafiltration membrane with a molecular weight cut-off of 10 kDa to effectively retain the silk fibroin protein and allow the papain to pass through. Place the ultrafiltration membrane containing the residual protein in a centrifuge tube and repeat the following operations 3 times: add 1 - 2 ml of PBS, use an ultrasonic disruptor at a power of 20%, in pulse mode, for 30 seconds, centrifuge at 8000 rpm for 2 minutes, collect the solution, combine the solutions collected 3 times, and obtain silk fibroin protein after drying.

10. The silk fibroin protein composite gel according to claim 9, wherein the water bath is at 65 °C.

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