Bionic regenerated silk protein multifunctional hydrogel suture line as well as preparation method and application thereof

Abstract

The invention discloses a bionic regenerated silk protein multifunctional hydrogel suture line and a preparation method thereof, and belongs to the field of biomedical materials. The suture line is composed of an inner core layer and an outer shell layer, the inner core layer is composed of 12 strands of regenerated fibroin hydrogel fibers adsorbing antibacterial zinc ions, and the outer shell layer is composed of methacrylic acid sericin loaded with procaine. The preparation method comprises the following steps: extracting regenerated silk fibroin from silkworm cocoons, preparing a spinning solution, introducing zinc ion coordination and orientation traction through wet spinning to prepare high-strength regenerated silk fibroin hydrogel fibers, twisting the fibers into threads after the fibers adsorb zinc ions, soaking the threads in a methacrylic acid sericin solution loaded with procaine, and drying the threads to obtain the high-strength regenerated silk fibroin hydrogel fibers. And gelatinizing and sterilizing to obtain the hydrogel suture line. The suture line is prepared on the basis of hydrogel fibers which are prepared through orientation traction and zinc ion crosslinking and have better mechanical properties, has the advantages of being simple and easy to implement, low in cost and capable of being produced on a large scale, and has the excellent mechanical properties, good biocompatibility and multiple functions of easing pain, resisting bacteria and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials, and in particular relates to a biomimetic regenerated silk protein multifunctional hydrogel suture thread, a preparation method thereof, and an application thereof. Background Art

[0002] Surgical sutures are a time-honored and indispensable medical product used to close damaged tissue and promote wound healing. Despite the recent development of alternatives such as bio-glue, adhesives, and nails, sutures still dominate due to their high stability, universal applicability, and ease of manipulation. Silkworm cocoons, produced by the silkworm Bombyx mori, are primarily composed of silk fibroin (70%-75%) and sericin (20-25%). Fibroin acts as an internal reinforcing fiber, providing mechanical strength, while sericin serves as an outer matrix, maintaining structural integrity. The complex structure of silk fibroin imparts excellent mechanical properties, adjustable biodegradability, good biocompatibility, low immunogenicity, and processability. These unique characteristics offer new applications for silk fibroin in areas such as tissue engineering, drug delivery, regenerative medicine, medical devices, biosensors, and bioprinting.

[0003] The mechanical properties of sutures are key indicators to ensure the success of the operation, including tensile strength, knot strength, elongation at break and creep resistance. Especially in high-strength demand scenarios such as cardiovascular suture and orthopedic tendon repair, sutures need to withstand dynamic mechanical loads and maintain structural stability for a long time. Silk sutures are mainly prepared based on silk fibroin fibers, and their mechanical properties mainly depend on the mechanical properties of silk fibroin fibers. Although silk fibroin fibers themselves have a high theoretical strength (about 500MPa), in the actual preparation of silk fibroin fibers, due to defects in the preparation process such as repeated pulling, the mechanical strength, flexibility and structural stability of silk fibroin fibers are easily poor, resulting in the actual mechanical properties of silk sutures (usually tensile strength <300MPa) being difficult to meet the needs of complex surgeries.

[0004] In addition to having good mechanical properties, ideal sutures must also have antibacterial properties (preventing postoperative infection), analgesic and anti-inflammatory effects (reducing tissue reaction, anti-inflammatory and reducing pain) and active repair functions (promoting cell adhesion and tissue regeneration). Wound infection is a common complication in surgical operations, manifested as local or systemic infection caused by bacterial colonization. Although antibiotics can reduce the risk of infection, the emergence of multidrug-resistant bacteria has reduced their effectiveness year by year. Pain is an issue worthy of attention. Postoperative pain (especially severe pain from skin incisions) reaches its peak on the first day after surgery. Persistent pain can affect the patient's vital signs, leading to shortness of breath, increased blood pressure, insomnia and decreased immunity. Analgesics are often used clinically for postoperative analgesia, but systemic use of analgesics is prone to drug resistance and adverse reactions.

[0005] Existing sutures usually combine analgesic and anti-inflammatory drugs with silk protein to prepare sutures with analgesic and anti-inflammatory effects. For example, patent CN106729944A discloses a silk protein suture with analgesic and anti-inflammatory effects. The suture is prepared by combining natural Chinese herbal medicine, plant extracts and silk protein. Although this suture has analgesic and anti-inflammatory effects, it does not mention wound infection and preventive effects. Patents CN105457084A and CN105999376A respectively introduce a medical suture with both analgesic and antibacterial functions, but the degradation products of polymer sutures usually contain lactic acid, which may cause local inflammation and soreness and other discomfort reactions, thereby affecting wound healing. In addition, compared with natural silk sutures, polymer sutures have a complex production process and high production costs, making them difficult to popularize. Summary of the Invention

[0006] In response to the shortcomings and deficiencies of the aforementioned prior art, the present invention provides a method for preparing a biomimetic regenerated silk protein multifunctional hydrogel suture. Based on regenerated silk fibroin and sericin, antibacterial regenerated silk fibroin hydrogel fibers are prepared using an improved wet spinning process that incorporates zinc ion coordination crosslinking, directional traction, and zinc ion loading. The fibers are then twisted into a suture, which is then coated with procaine-loaded methacryloyl sericin to produce a biomimetic regenerated silk protein multifunctional hydrogel suture.

[0007] This study uses an improved wet-spinning process to successfully produce a high-strength, antibacterial regenerated silk fibroin hydrogel fiber with zinc ion-assisted cross-linking. Sutures prepared from this fiber through a simple twisting process exhibit excellent mechanical properties and broad-spectrum antibacterial activity, significantly reducing the risk of bacterial resistance. Furthermore, the porous methacryloyl-coated sericin used as the outer drug-carrying coating of the suture enables the sustained release of procaine and zinc ions, thereby imparting long-lasting analgesic and antibacterial properties. The suture also exhibits good cytocompatibility and histological compatibility, demonstrating significant potential for clinical translation.

[0008] To achieve the above object, the present invention first provides a method for preparing a biomimetic regenerated silk protein multifunctional hydrogel suture, comprising the following steps:

[0009] (1) Extraction of regenerated silk fibroin: natural silk cocoons are washed, dried, degummed, dried, dissolved, dialyzed, filtered, centrifuged, and freeze-dried to obtain silk fibroin;

[0010] (2) preparing regenerated silk fibroin hydrogel fibers by wet spinning: dissolving the silk fibroin obtained in step (1) in formic acid to prepare a spinning solution, pumping the spinning solution into a methanol coagulation bath through a hose using a microfluidic pump, and orienting and pulling the prepared fibers using a multifunctional mechanical tester. The pulled fibers are immersed in a recrystallization bath for recrystallization and solidified and dried to obtain high-strength silk fibroin hydrogel fibers;

[0011] (3) Antibacterial treatment: soaking the high-strength silk fibroin hydrogel fiber obtained in step (2) in an antibacterial solution, and after the solution is clarified, taking out the fiber and sterilizing it to obtain a high-strength, antibacterial regenerated silk fibroin hydrogel fiber;

[0012] (4) Preparation of suture: twisting the high-strength, antibacterial regenerated silk fibroin hydrogel fibers obtained in step (3) into suture;

[0013] (5) Preparing a light-cured methacryloyl sericin solution: dissolving sericin powder in a phosphate buffer solution (pH = 9.5), adding methacrylic anhydride in a dark environment, and reacting for 12 to 72 hours to obtain a methacryloyl-modified sericin solution, dialyzing, and freeze-drying to obtain the methacryloyl-modified sericin; preparing a methacryloyl-modified sericin solution with a mass concentration of 5%, mixing the methacryloyl-modified sericin solution with a photoinitiator solution in a dark environment, and obtaining a light-cured methacryloyl sericin solution;

[0014] (6) Assembly of biomimetic silk protein multifunctional hydrogel suture: soak the suture in a procaine-loaded methacryloyl sericin solution for 2-3 minutes, photocuring it into gel under ultraviolet light, remove the suture and sterilize it to obtain a biomimetic silk protein multifunctional hydrogel suture.

[0015] In one embodiment of the present invention, in step (1), the specific process of washing, drying, degumming and drying is to cut the natural silk cocoons into pieces, wash them with deionized water for 4-5 times and dry them, take the washed and dried silk cocoons and place them in a Na2CO3 solution, boil them for 1 hour to degumming, wash them with deionized water for 4-5 times after degumming, and dry them, wherein the mass volume ratio of silk cocoons to Na2CO3 solution is 25-35g / 10-14L, and the concentration of the Na2CO3 solution is 0.02-0.025mol / L.

[0016] In one embodiment of the present invention, in step (1), the dissolution process is specifically as follows: taking the degummed and dried silk cocoons and dissolving them in a lithium bromide solution with a concentration of 9-9.4 mol / L, stirring and dissolving them in a 60°C water bath for 4-6 hours, wherein the mass volume ratio of the degummed and dried silk cocoons to the lithium bromide solution is 25-27 g:90-100 mL.

[0017] In one embodiment of the present invention, in step (1), the dialysis process is specifically as follows: after the cocoons are completely dissolved, they are cooled to room temperature and dialyzed for 48-72 hours, with deionized water being replaced at least 3 times every 24 hours; the molecular weight retained during dialysis is 3500Da.

[0018] In one embodiment of the present invention, in step (1), the filtration, centrifugation, and freeze-drying process specifically comprises filtering the dialyzed primary extract through a nylon filter cloth (500 mesh), centrifuging at 5000 r / min, and freeze-drying the supernatant. The freeze-dried product must be stored at -80°C.

[0019] In one embodiment of the present invention, in step (2), the concentration of the spinning solution is 12-15%.

[0020] In one embodiment of the present invention, in step (2), the spinning solution is pumped into the methanol coagulation bath at a speed of 0.5 to 1 mL / min, and the diameter of the hose is 200 μm.

[0021] In one embodiment of the present invention, in step (2), the spinning solution is immersed in a 95% methanol coagulation bath for 10 to 30 minutes.

[0022] In one embodiment of the present invention, in step (2), the methanol coagulation bath further contains zinc ions, and the concentration of zinc ions is 0.5-5%, preferably 0.5-4.5%, more preferably 1-4.5%, and even more preferably 1.5-3%.

[0023] In one embodiment of the present invention, in step (2), the methanol coagulation bath is a mixed solution of zinc chloride and 95% methanol, and the mass concentration of the zinc chloride is 0.5-5%.

[0024] In one embodiment of the present invention, in step (2), the ambient temperature during the orientation traction is 23-25°C, the air humidity is 50%, the traction speed is v=200mm / min, and after the orientation traction, the tension is fixed with a force of 1N for 2-3 minutes.

[0025] In one embodiment of the present invention, in step (2), the traction ratio of the orientation traction is 4 to 8 times, preferably 5 to 7 times, and more preferably 6 times.

[0026] In one embodiment of the present invention, in step (2), the solution of the recrystallization bath is 75% ethanol solution, the time the fiber is in the recrystallization bath is 10 minutes, the curing temperature is 25-37° C., and the curing time is 10-30 minutes.

[0027] In one embodiment of the present invention, in step (2), the solution in the recrystallization bath further contains zinc ions, and the concentration of the zinc ions is 0.5-5%, preferably 0.5-4.5%, more preferably 1-4.5%, and even more preferably 1.5-3%.

[0028] In one embodiment of the present invention, in step (2), the recrystallization bath is a mixed solution of zinc chloride and 75% ethanol, and the mass concentration of the zinc chloride is 2.5%.

[0029] In one embodiment of the present invention, in step (3), the antibacterial solution is an aqueous solution of zinc chloride, the immersion time is 4 to 6 hours, and the mass concentration of zinc chloride in the solution is 0.1 to 5%, preferably 0.5 to 5%, more preferably 1 to 4%, and even more preferably 1.5 to 3.5%.

[0030] In one embodiment of the present invention, the sterilization treatment is high temperature and high pressure sterilization, the temperature during sterilization is 121°C-134°C, the pressure is 1.02-2.03 kg / cm 2 , the sterilization time is 30min-60min.

[0031] In one embodiment of the present invention, in step (4), the specific process of twisting into a suture thread is to twist the high-strength, antibacterial regenerated silk fibroin hydrogel fiber in a Z-shape with a twist of 1 to 12 twists / cm, dry and fix, and the twist is preferably 2 to 10 twists / cm, more preferably 3 to 9 twists / cm, and even more preferably 5 to 9 twists / cm.

[0032] In one embodiment of the present invention, in step (5), the mass volume ratio of sericin powder to phosphate buffer is 0.8-1 g:4-5 mL, and the mass ratio of methacrylic anhydride to sericin is 3:5.

[0033] In one embodiment of the present invention, in step (5), dialysis is performed in a dialysis bag with a molecular weight cut-off of 3500 Da, the dialysis time is 48 to 72 hours, and the water is changed 2 to 3 times a day.

[0034] In one embodiment of the present invention, in step (5), the photoinitiator is Irgacure 2959, the concentration of the photoinitiator solution is 0.01 g-0.02 / mL, and the volume ratio of the methacrylated sericin solution to the photoinitiator solution is 100-1000:1, preferably 100:1 or 1000:1.

[0035] In one embodiment of the present invention, in step (6), the method for preparing the procaine-loaded methacryloyl sericin solution is to disperse procaine in the methacryloyl sericin solution, and the mass volume ratio of procaine to methacryloyl sericin solution is 2.5-5 g:100 mL.

[0036] In one embodiment of the present invention, in step (6), the photocuring into glue under ultraviolet light has a wavelength of 365 nm and an irradiation time of 1-2 min.

[0037] In one embodiment of the present invention, in step (6), the sterilization treatment is high temperature and high pressure sterilization, the temperature during sterilization is 121°C-134°C, and the pressure is 1.02-2.03 kg / cm 2 , the sterilization time is 30min-60min.

[0038] The present invention also provides a bionic regenerated silk protein multifunctional hydrogel suture thread prepared according to the above preparation method.

[0039] The present invention also provides applications of the biomimetic regenerated silk protein multifunctional hydrogel suture in the fields of biomedicine and tissue engineering.

[0040] Beneficial effects:

[0041] 1. The present invention first prepares silk fibroin by dissolving, dialyzing, and freeze-drying degummed silk cocoons. The silk fibroin is then wet-spun, oriented, drawn, and recrystallized to produce regenerated silk fibroin hydrogel fibers with both strength and toughness. After antibacterial treatment, the fibers are twisted to form sutures, which are then immersed in a light-cured methacryloyl sericin solution to produce a multifunctional hydrogel suture. As the outer coating of the suture, the methacryloyl sericin exhibits a unique loose and porous structure, providing an ideal platform for drug delivery, thereby exerting long-lasting effects during tissue repair and regeneration, and offering a promising approach for the preparation of long-lasting functional sutures.

[0042] 2. The mechanical properties of the suture of the present invention primarily rely on the mechanical properties of the regenerated silk fibroin hydrogel fibers. By vertically orienting the fibers formed after coagulation in a coagulation bath, this directional orientation can, on the one hand, induce the molecular chains and other structures in the fibers to align axially through physical effects, resulting in a dense internal structure of the fibers, increased fiber crystallinity, and induced β-folding, thereby greatly improving the mechanical properties of the fibers. On the other hand, the method of first orienting and then re-crystallizing the fibers can prolong the recrystallization curing time, further contributing to the improvement of the mechanical properties of the fibers.

[0043] 3. When preparing the hydrogel fiber used in the suture of the present invention, a certain amount of zinc chloride is added to the coagulation bath and the recrystallization bath. When the fiber is in the coagulation bath and recrystallization, the zinc ions can coordinate and cross-link with the serine of the silk fibroin to form an ionic covalent bond. The cross-linking of the zinc ions can further improve the mechanical properties of the fiber. At the same time, the embedding of zinc ions also gives the fiber good antibacterial properties. After the high-strength regenerated silk fibroin hydrogel fiber after recrystallization and curing is immersed in an antibacterial solution for a period of time, the antibacterial ions can be adsorbed on the surface of the fiber, achieving a synergistic improvement in the high strength and antibacterial properties of the fiber, while meeting the high requirements of biomedical materials for mechanical properties and biocompatibility.

[0044] 4. This invention utilizes an improved wet-spinning technique to produce high-strength regenerated silk fibroin hydrogel fibers. The introduction of zinc ions enhances the fiber's mechanical properties. Furthermore, a multi-strand twisting process further optimizes the fiber's mechanical properties, resulting in the successful production of a hydrogel suture with both excellent mechanical strength and biocompatibility. This suture not only meets the mechanical performance requirements for medical applications but also offers a potential functional solution for tissue repair and regenerative medicine, providing important technical support for the development of high-performance medical sutures.

[0045] 5. The present invention loads zinc ions for antibacterial effects. Compared to antibiotics, zinc ions are less likely to develop drug resistance. Furthermore, as an essential element for the human body, zinc ions participate in the synthesis of multiple coenzymes, which is beneficial for wound healing. Procaine is a commonly used local anesthetic in clinical practice, with a rapid onset of action, stable effects, and safety. The present invention has a simple preparation process, and the product thickness, structure, and length are highly adjustable. No large amounts of toxic and harmful reagents are used in the preparation of the sutures, thus protecting human health and making it widely applicable in the medical and health fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the preparation method of the biomimetic regenerated silk protein multifunctional hydrogel suture thread of the present invention;

[0047] Figure 2 Schematic diagram of the drawing method (A) in the embodiment and experiments ① to ② of the present invention and the wet spinning method in experiment ③ (B);

[0048] Figure 3 The morphological characteristics (A) and diameters (B) of STZS sutures with different strand numbers;

[0049] Figure 4 Mechanical characterization of sutures with different twists and PSTZS 12 、STZS 12 , 5-0 Mechanical properties evaluation;

[0050] Figure 5 TS for suture line 6 / 12、TZS 6 / 12 、STZS 6 / 12 Mechanical properties evaluation;

[0051] Figure 6 Schematic diagram of suture knot tension mechanical performance test (A) and knot tension mechanical performance results (B);

[0052] Figure 7 TZS suture 12 、STZS 12 、PSTZS 12 Scanning electron microscope images of

[0053] Figure 8 SerMA, natural regenerated silk fibroin and procaine (A) and suture PSTZS 12 、STZS 12 、TZS 12 TS 12 (B) FTIR analysis results;

[0054] Figure 9 A shows the results of live and dead cell staining; B and E show the results of 24-hour fibroblast migration assay and statistical analysis; C and D show the results of CCK-8 and drug release assay, respectively;

[0055] Figure 10 The results of in vivo biocompatibility experiments in rats;

[0056] Figure 11 The results of the E. coli antibacterial test and adhesion test;

[0057] Figure 12 The results of the antibacterial test and adhesion test on Staphylococcus aureus are shown;

[0058] Figure 13 A is a schematic diagram of the skin incision model and sampling time, B is the suture and healing effect of the skin incision, C is the H&E staining results of the skin incision (1, 3, 5, 7, 10, 14 days); D is the STZS 12 、PSTZS 12 HE staining results of organs 28 days after subcutaneous implantation. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0060] In order to better understand the present application, the present application is further described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments described in the specific embodiments are only part of the embodiments of the present application and do not limit the scope of protection of the present application.

[0061] Unless otherwise specified in the examples, the experiments were conducted under conventional conditions or manufacturer recommendations. Reagents and instruments used without manufacturer identification are commercially available. The sericin powder used in the examples and comparative examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0062] Explanation of the English abbreviations corresponding to fibers and sutures:

[0063] RSF-Zn 2+ Crosslinked (drawn): When preparing hydrogel fibers, zinc chloride is added to both the coagulation bath and the recrystallization bath, and orientation drawing is required after the coagulation bath.

[0064] RSF-Zn 2+ Crosslinked: When preparing hydrogel fibers, zinc chloride is added to both the coagulation bath and the recrystallization bath, but no orientation pulling is performed after the coagulation bath.

[0065] RSF (drawn): When preparing hydrogel fibers, zinc chloride is not added to the coagulation bath and the recrystallization bath, but orientation drawing is required after the coagulation bath.

[0066] RSF: When preparing hydrogel fibers, no zinc chloride is added to the coagulation bath and the recrystallization bath, and no orientation drawing is performed after the coagulation bath.

[0067] TS: When preparing silk fibroin hydrogel fibers, neither the coagulation bath nor the recrystallization bath contained zinc ions, the fibers were not treated with antibacterial agents, and the twisted sutures were not immersed in a light-cured methacryloyl sericin solution containing procaine. x The x in the equation represents the number of fiber strands in the suture, and the values ​​of x are 2, 4, 6, 8, 10, 12, and 14.

[0068] TZS: When preparing silk fibroin hydrogel fibers, the coagulation bath and recrystallization bath contained 2.5% zinc ions, and the twisted sutures were not immersed in a light-cured methacryloyl sericin solution containing procaine. x The x in the equation represents the number of fiber strands in the suture, and the values ​​of x are 2, 4, 6, 8, 10, 12, and 14.

[0069] STZS: When preparing silk fibroin hydrogel fibers, the coagulation bath and recrystallization bath contain 2.5% zinc ions, and the twisted sutures are immersed in a light-cured methacryloyl sericin solution without procaine.x The x in the equation represents the number of fiber strands in the suture, and the values ​​of x are 2, 4, 6, 8, 10, 12, and 14.

[0070] PSTZS: When preparing silk fibroin hydrogel fibers, the coagulation bath and recrystallization bath contain 2.5% zinc ions, and the twisted sutures are immersed in a light-cured methacryloyl sericin solution containing procaine. x The x in the equation represents the number of fiber strands in the suture, and the values ​​of x are 2, 4, 6, 8, 10, 12, and 14.

[0071] PSTS: When preparing silk fibroin hydrogel fibers, neither the coagulation bath nor the recrystallization bath contains zinc ions, and the fibers are not subjected to antibacterial treatment. The twisted sutures are immersed in a light-cured methacryloyl sericin solution containing procaine.

[0072] The mechanical properties of the hydrogel fiber were tested by taking a 10 cm long hydrogel suture sample and performing a uniaxial tensile test at a stretching speed of 20 mm / min until the fiber broke, and recording the stress and strain at the time of breakage.

[0073] The suture knot tension test method involves tying a square knot (the knot is located at the midpoint of the suture) with each suture set to an initial length of 10 cm. Tension is applied at a rate of 20 mm / min until the suture breaks. (A valid test is considered if the breakage distance from the knot is ≥1 cm.) The breaking force and maximum strain are recorded to assess the knot strength of the suture in surgical applications.

[0074] Drug release, migration testing, in vitro and in vivo biocompatibility

[0075] Drug release test method: Prepare two 40 cm long PSTZS 12 The sutures were soaked in 5 mL of phosphate buffered saline (PBS) and placed in a constant temperature environment at 37° C. Every 24 hours, 2 mL of the solution was taken out as a sample and immediately replenished with the same volume of PBS buffer. The entire test lasted for 7 days.

[0076] The absorption peak of zinc ion was measured at a wavelength of 620 nm using a spectrophotometer, and the absorption peak of procaine was measured at a wavelength of 290 nm. In order to accurately quantify the release of the drug, standard solutions of procaine and zinc ion were prepared respectively, with concentrations of 1 g / 1 mL, 0.5 g / mL, 0.25 g / mL, and 0.125 g / mL, and a standard curve was established using these standard solutions. Based on the standard curve and the measured absorbance (OD value), the drug concentration released per day was calculated. The cumulative release percentage of the drug was calculated according to the following formula to evaluate the release of PSTZS 12Sustained release of procaine and zinc ions by sutures under simulated physiological conditions.

[0077]

[0078] Among them C x (mg / mL) represents the drug concentration released after x days, and T (mg / mL) represents the total drug loading.

[0079] In vitro biocompatibility test

[0080] Biocompatibility experiments were performed using CCK-8 kit and calcein / PI cell viability / cytotoxicity assay kit. The experiments followed the ISO 10993-12 standard and weighed 1 gram of sterilized STZS. 12 and PSTZS 12 The initial determination of the amount of DMEM culture medium absorbed by the sample was 2 ml. Subsequently, 1 gram of sterilized STZS was weighed again. 12 and PSTZS 12 The samples were added to 12 ml of DMEM medium, sonicated for 1 hour to ensure thorough mixing, and then incubated at 37°C for 24 hours. After the incubation, the supernatant was aspirated and filtered through a 0.22 μm filter to prepare the extract.

[0081] L929 fibroblasts were cultured at 4 × 10 3 Cells were seeded at a density of 100 cells / mL in a 96-well plate, and 100 μL of the cell suspension was added to each well. 10 μL of the above extract was added to the experimental wells, while 10 μL of PBS buffer was added to the control wells. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0082] According to the manufacturer's instructions, CCK-8 reagent and calcein / PI reagent were added to the wells after 24, 48, and 72 hours of culture, respectively. Cell viability and cytotoxicity were detected by spectrophotometer and inverted fluorescence microscope, respectively, to evaluate the activity of STZS. 12 and PSTZS 12 Effects on cell growth and survival.

[0083] 24h fibroblast migration assay

[0084] According to ISO 10993-12, 1 gram of sterilized STZS was weighed. 12 and PSTZS 12 The initial determination of the amount of DMEM culture medium absorbed by the sample was 2 ml. Subsequently, 1 gram of sterilized STZS was weighed again. 12 and PSTZS 12, were added to 12 ml of DMEM medium, sonicated for 1 hour to ensure thorough mixing, and then incubated at 37°C for 24 hours. After the incubation, the supernatant was aspirated and filtered through a 0.22 μm filter membrane to prepare an extract.

[0085] Then, take a six-well plate and draw three parallel horizontal lines along the ruler on the bottom of the plate with a marker as marking lines. Add 2 ml of L929 fibroblast suspension (cell density 4×10 3 Cells were seeded into each well of a six-well plate (100 cells / ml) and incubated in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to adhere and grow. Once the cells were confluent, three vertical lines were drawn perpendicularly between the well plate and the marked line using a 200-μl pipette tip, ensuring that the lines intersected the marked line to create a clear scratched area.

[0086] Remove the old culture medium and gently rinse the plate 3-4 times with PBS buffer until the cells are completely washed away. Add DMEM culture medium, STZS 12 Extract or PSTZS 12 Extract. Take a 10× magnification photograph using a microscope, which serves as a 0-hour control. Subsequently, return the plate to a 37°C, 5% CO2 incubator and continue incubating for 24 hours. Observe the width of the scratch at the same location under a microscope and take a photograph.

[0087] Finally, the captured images were analyzed and processed using ImageJ software, and STZS was evaluated by measuring the changes in scratch width. 12 and PSTZS 12 Effects of the extract on cell migration ability.

[0088] In vivo biocompatibility and organ toxicity

[0089] The animal experiments were approved by the Animal Ethics Committee of Jiangnan University, China (approval number: JN.No20240630S0800915

[372] ). SPF-grade Sprague-Dawley rats (male, 6-8 weeks old, weighing 200-220 g, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., China) were used in the experiments. A 2 cm long skin incision was made on the back of the rats, and subcutaneous expansion was performed around the incision. Subsequently, a 2 cm long STZS was implanted. 12 or PSTZS 12 All operations were performed under sterile conditions.

[0090] The rats were killed on the 3rd, 7th, 14th and 28th days after surgery, and the sutures and surrounding tissues were collected. On the 28th day, the normal rats, STZS 12 、PSTZS 12Heart, liver, spleen, lung, and kidney specimens were collected and fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE).

[0091] Antibacterial performance test

[0092] Staphylococcus aureus (S. aureus ATCC 25923) and Escherichia coli (E. coli ATCC 25922) were cultured separately. TS 12 、STS 12 、TZS 12 、STZS 12 and PSTZS 12 The sutures were soaked in 5 ml of sterile PBS buffer and placed in a shaker at 37°C. 2 ml of the solution was taken out as a sample every 24 hours and replenished with 2 ml of sterile PBS buffer for 3 days.

[0093] Use the bacterial stick to dip the bacteria into the solution with a concentration of 1×10 5 Staphylococcus aureus and Escherichia coli at 500 CFU / mL were evenly inoculated on LB medium plates. Every day, 10 microliters of the 2 ml sample extract was taken and dropped onto a sterile blank drug-sensitive paper disc (6 mm in diameter). The drug-sensitive paper disc was placed on the bacterial plate and then incubated in a 37°C, 5% CO2 incubator for 12 hours. After the incubation period, a vertical tangent was drawn from the edge of the inhibition zone to the edge of the drug-sensitive paper, and the distance was measured using ImageJ software to evaluate the antibacterial effect of the suture.

[0094] Bacterial adhesion assay

[0095] Cut 2 cm long 5-0 TS 12 、STS 12 、TZS 12 、STZS 12 and PSTZS 12 Sutures were placed on a plate inoculated with Staphylococcus aureus and Escherichia coli and incubated in a 37°C, 5% CO2 incubator for 24 hours. After incubation, the sutures were removed from the plate, fixed in a 4% paraformaldehyde solution for 2 hours, and gently rinsed three times in deionized water. The samples were then freeze-dried and observed for bacterial attachment using a scanning electron microscope (SEM).

[0096] Skin incision suture experiment

[0097] SD rats were anesthetized by intraperitoneal injection of tribromoethanol (25 mg / kg). After anesthesia, the rats were fixed in a prone position and a 2 cm long and 2 mm deep dermal incision was made on the back skin. STZS12 and PSTZS 12 The incision was sutured with sutures. The healing status of the incision was observed on days 1, 2, 5, 7, 10, and 14 after surgery. Skin suture tissue samples were collected on days 1, 3, 5, 7, 10, and 14 after surgery for HE staining. All samples were fixed with 4% paraformaldehyde for 24 hours, then dehydrated and embedded in paraffin. 4-micron thick sections were cut from the embedded tissue blocks for hematoxylin-eosin (HE) staining. Incision suture experiments were performed on the back of rats, with 5-0 STZS 12 and PSTZS 12 Sutures were performed and the healing process was monitored on postoperative days 1, 3, 5, 7, 10, and 14.

[0098] Example 1

[0099] A method for preparing a biomimetic regenerated silk protein multifunctional hydrogel suture thread, the process is as follows Figure 1 As shown, the following steps are included:

[0100] 1. Preparation of high-strength regenerated silk fibroin hydrogel fibers:

[0101] (1) Cocoon degumming: 35 g of cleaned and dried silk cocoons were placed in 14 L of 0.02 M Na2CO3 solution and boiled for 1 hour to degummed. After degumming, the cocoons were washed 4-5 times with deionized water and dried.

[0102] (2) Extraction of regenerated silk fibroin: 27 g of degummed and dried silk cocoons were weighed and dissolved in 100 mL of 9.3 mol / L lithium bromide solution, and stirred in a 60°C water bath for 6 hours. After the cocoons were dissolved, they were cooled to room temperature and dialyzed (molecular weight cutoff 3500 Da) for 72 hours, with deionized water replaced at least 3 times every 24 hours. The solution was filtered through a nylon filter membrane (500 mesh) and centrifuged at 5000 r / min. The supernatant was freeze-dried to obtain regenerated silk fibroin.

[0103] (3) Preparation of regenerated silk fibroin hydrogel by wet spinning: 1.5 g of the regenerated silk fibroin obtained in step (2) was weighed and dissolved in 10 mL of formic acid to prepare a spinning solution with a concentration of 15%. The spinning solution was passed through a 200 μm hose at a speed of V = 0.5 mL / min and extruded into a zinc chloride-methanol coagulation bath solution with a mass concentration of 2.5%, and soaked for 10 minutes to obtain regenerated silk fibroin hydrogel fibers. The zinc chloride-methanol coagulation bath solution was prepared by mixing 2.5 g of zinc chloride and 100 mL of 95% methanol.

[0104] (4) Orientation pulling and recrystallization solidification: The regenerated silk fibroin hydrogel fiber obtained in step (3) was taken out and oriented and pulled 6 times at v = 200 mm / min on a multifunctional mechanical tester at an ambient temperature of 23°C and an air humidity of 50%. The pulling process was as follows: Figure 2 As shown in A, the tension was fixed with a force of 1 N for 2 minutes; the hydrogel fiber after traction was immersed in a zinc chloride ethanol recrystallization bath solution with a mass concentration of 2.5% for 30 minutes, recrystallized and solidified, and dried at a drying temperature of 37°C and a drying time of 5 minutes to obtain high-strength regenerated silk fibroin hydrogel fiber. The zinc chloride ethanol recrystallization bath solution was composed of a mixture of 2.5g zinc chloride and 100mL 75% ethanol.

[0105] 2. Preparation of high-strength antibacterial silk fibroin hydrogel fibers:

[0106] Take 5 g of the high-strength regenerated silk fibroin hydrogel fiber obtained in step 1, place it in 100 mL of 2.5% zinc chloride aqueous solution, stir for 6 hours until the solution is clear, take it out and sterilize it at high temperature and high pressure (121°C) for 30 minutes to obtain high-strength antibacterial silk fibroin hydrogel fiber.

[0107] 3. Fiber twisting into suture: Take 12 strands of fiber about 60 cm long, twist them in a Z-shape with a twist of 8 twists / cm, and fix them with a tension of 4 N to obtain a suture.

[0108] 4. Preparation of light-cured methacryloyl sericin solution:

[0109] At 35°C, 6 g of sericin powder was dissolved in 30 mL of phosphate buffer (pH 9.5). Protect from light throughout the following steps. 3.6 g of methacrylic anhydride was added to 50 mL of phosphate buffer and stirred at 50°C for 24 hours to obtain a methacrylic anhydride solution. 30 mL of sericin solution was injected into the methacrylic anhydride solution using a microfluidic pump at a rate of 5 mL / min. The mixture was stirred in a 50°C water bath for 24 hours. The solution was then dialyzed in a dialysis bag (MWCO = 3500 Da) for 2-3 days, with the water changed 2-3 times daily. A 5% sericin solution was prepared. Next, 1 g of the photoinitiator Irgacure 2959 was dispersed in 100 mL of deionized water to obtain an Irgacure 2959 solution. The methacrylated sericin solution and the Irgacure 2959 solution were thoroughly mixed at a ratio of 100:1 by volume and stored at 4°C in the dark to obtain a light-cured methacryloyl sericin solution.

[0110] 5. Assembly of biomimetic silk protein multifunctional hydrogel suture: Add 5g of procaine to 100mL of light-cured methacryloyl sericin solution and stir at 37°C for 10min. Place the twisted suture in a light-cured sericin solution containing 5% procaine, soak for 2min, take out and irradiate with ultraviolet light (wavelength 365nm, lasting 1 minute), and then soak in the light-cured sericin solution. Repeat this operation 2-3 times. After the operation is completed, autoclave for 30min to prepare the biomimetic silk protein multifunctional hydrogel suture PSTZS. 12 .

[0111] Example 2

[0112] The difference between Example 2 and Example 1 is that the coagulation bath solution in step (3) is 95% methanol solution, and the ethanol recrystallization bath in step (4) is 75% ethanol. Steps 2, 4, and 5 are omitted, and the suture is named TS 12 .

[0113] Before preparing sutures, the effects of different spinning solution concentrations (8%, 12%, 15%, and 18%) on the mechanical properties of silk fibroin hydrogel fibers without traction and antibacterial treatment were investigated. The results showed that as the spinning solution concentration increased from 8% to 15%, the breaking stress of the hydrogel fibers increased from 5.93±1.77MPa to 20.90±3.07MPa. At 8%, 12%, and 15% spinning solution concentrations, the breaking strains of the fibers were 102.60±21.61%, 191.80±15.35%, and 177.10±18.97%, respectively. As the spinning solution concentration increased to 18%, the breaking stress of the fibers decreased to 12.74±1.63MPa, and the breaking strain was 213.40±12.00%. Therefore, when the spinning solution concentration is 15%, the mechanical properties of the fiber are the best. When the spinning solution concentration increases to 18%, due to its high viscosity, it is easy to cause clogging of the gun tip, resulting in uneven fibers, which in turn affects its mechanical properties.

[0114] The effects of zinc ion concentration in the coagulation and recrystallization baths on the breaking stress of undrawn and unantibacterial treated fibers were similar to those of spinning solution concentration. As the zinc ion concentration increased from 0.5% to 5%, the breaking stresses were 20.13±0.54MPa, 21.42±0.10MPa, 28.61±3.25MPa, and 10.48±1.18MPa, respectively, and the breaking strains were 299.90±64.52%, 266.80±11.95%, 277.10±53.29%, and 170.70±71.95%, respectively. The results showed that a zinc ion crosslinking concentration of 2.5% was optimal. A concentration too low (e.g., 1%) resulted in incomplete crosslinking, while a high concentration (5%) increased fiber brittleness, affecting mechanical properties.

[0115] After determining the optimal spinning solution concentration and zinc ion concentration, different regenerated silk fibroin hydrogel fibers were prepared using different methods. The specific hydrogel fibers prepared are as follows:

[0116] ① According to the method of step 1 in Example 1, regenerated silk fibroin hydrogel fibers were prepared and named RSF-Zn 2+ The mechanical properties of the hydrogel fibers at different pulling ratios of 0, 2, 4, 6 and 8 were studied.

[0117] ② Regenerated silk fibroin hydrogel fibers were prepared according to the method of step 1 in Example 2. The fibers were named RSF (drawn), and the mechanical properties of the hydrogel fibers at different drawing magnifications of 0, 2, 4, 6, and 8 were studied.

[0118] ③ Silk fibroin hydrogel fibers were prepared according to a conventional pulling method. The difference between this method and Example 2 is that steps (3) and (4) are as follows:

[0119] Preparation of regenerated silk fibroin hydrogel by wet spinning: 1.5 g of regenerated silk fibroin obtained in step (2) was weighed and dissolved in 10 mL of formic acid to prepare a spinning solution with a concentration of 15%. The spinning solution was passed through a 200 μm hose at a speed of V = 0.5 mL / min and squeezed into a 95% methanol coagulation bath solution containing 2.5% zinc chloride. After passing through a traction winding wheel 1 (ω1 = 20 r / min), it was immersed in ethanol (75%) containing 2.5% zinc chloride. It was stretched under the action of a traction winding wheel 2 (ω2 = 20 r / min) (the stretching ratio was 2 times). The traction process was as follows: Figure 2 As shown in B, the fibers collected by the winding wheel 2 are immersed in ethanol containing 2.5% zinc chloride for 30 minutes, taken out and dried to obtain regenerated silk fibroin hydrogel fibers.

[0120] Experiment ③ simultaneously studied the mechanical properties of the fibers at traction multiples of 0, 4, 6, and 8. The results of experiments ① to ③ are shown in Tables 1 to 3. Tables 1 and 2 show the diameter and mechanical properties of the hydrogel fibers at different traction multiples in experiments ① and ②. From the experimental results in Tables 1 and 2, it can be seen that as the traction intensity increases, the mechanical properties are more superior, but 8-fold traction will lead to a decrease in mechanical properties. However, excessive traction will cause the internal hydrogen bonds to break, affecting the mechanical properties. Compared with RSF (drawn), the mechanical properties of the fiber after zinc ion cross-linking are more excellent after traction. The optimal traction multiple is 6 times.

[0121] Table 1 Diameter and mechanical properties of hydrogel fibers in experiment ①

[0122]

[0123] Table 2 Diameter and mechanical properties of hydrogel fibers in experiment ②

[0124]

[0125]

[0126] In Experiment 3, zinc-crosslinked hydrogel fibers were prepared using a conventional drawing process. The diameter and mechanical properties of these fibers are shown in Table 3. A comparison of the data in Tables 1 and 2 shows that, compared to conventional drawing processes, the oriented drawing combined with recrystallization method of the present invention is more beneficial for improving the mechanical properties of hydrogel fibers. This is primarily because conventional drawing utilizes godet rollers and winding rollers for drawing, but requires controlling the roller speed and tension to draw the fibers. While this method enables integrated spinning, its effect on improving the mechanical properties of the fibers is relatively limited due to the insufficient coagulation and recrystallization time of the hydrogel fibers, and the fact that the drawing process is primarily driven by the centrifugal speed of the rollers, preventing the fibers from being oriented along the axial direction. Furthermore, fiber breakage and slippage are highly likely to occur during the spinning process, leading to uneven fiber drawing. The oriented drawing method of the present invention, through a step-by-step process, effectively avoids the inadequate fiber coagulation and recrystallization time. The directional oriented drawing physically induces the molecular chains and other structures in the fibers to align along the axial direction, resulting in a denser internal structure, increased fiber crystallinity, and the formation of β-sheets, significantly enhancing the mechanical properties of the fibers. The coordination cross-linking of zinc ions and serine in silk fibroin forms ionic covalent bonds, which, to a certain extent, improves the mechanical properties of wet-spun fibers.

[0127] Table 3 Diameter and mechanical properties of hydrogel fibers in experiment ③

[0128]

[0129] Example 3

[0130] The difference between Example 3 and Example 1 is that the light-cured sericin solution in step 5 does not contain procaine, and the suture is named STZS 12 .

[0131] The difference between Example 4 and Example 3 is that the number of fiber strands in step 3 is 6, and the suture is named STZS6.

[0132] Example 5

[0133] The difference between Example 5 and Example 3 is that the twist of the suture thread in step 3 is 1 Twist / cm.

[0134] Example 6

[0135] The difference between Example 6 and Example 3 is that the twist of the suture thread in step 3 is 2 Twists / cm.

[0136] Example 7

[0137] The difference between Example 7 and Example 3 is that the twist of the suture thread in step 3 is 4 Twists / cm.

[0138] Example 8

[0139] The difference between Example 8 and Example 3 is that the twist of the suture thread in step 3 is 12 Twists / cm.

[0140] Comparative Example 1

[0141] The difference between Comparative Example 1 and Example 1 is that steps 4 and 5 are omitted and the suture is named TZS 12 .

[0142] Comparative Example 2

[0143] Comparative Example 2 is commercial 5-0

[0144] Comparative Example 3

[0145] The difference between Comparative Example 3 and Comparative Example 1 is that the number of fiber strands in the suture is 6, and the suture is named TZS6.

[0146] Comparative Example 4

[0147] The difference between Comparative Example 4 and Example 2 is that the number of fiber strands in the suture is 6, and the suture is named TS6.

[0148] Comparative Example 5

[0149] The difference between Comparative Example 5 and Example 2 is that only steps 2 and 6 are omitted and the suture is named STS 12

[0150] Electron microscopy characterization of biomimetic regenerated silk fibroin hydrogel sutures with different strand numbers:

[0151] The freeze-dried biomimetic regenerated silk fibroin hydrogel suture was placed under a scanning electron microscope (SEM) to observe its microstructure. Figure 3 A shows that the light-cured methacryloyl sericin wraps the suture thread, and the twist is visible. Figure 3 B, STZS 12 The diameter is about 200 μm, and 5-0 Diameter 225μm equivalent, STZS 14 The diameter is 250μm.

[0152] Mechanical properties test results

[0153] Table 4 Suture TS 6 / 12 、TZS 6 / 12 、STZS 6 / 12 、PSTZS 12 , 5-0 Mechanical evaluation

[0154]

[0155]

[0156] Table 5 STZS with different twists 12 Mechanical evaluation of sutures

[0157]

[0158] Table 6 Suture knot tension mechanical evaluation

[0159]

[0160] Tables 4, 5, and 6 present the mechanical properties of different sutures, the mechanical properties of sutures with different twists, and the knot tensile strength, respectively. Table 4 shows that the addition of zinc ions to the coagulation and recrystallization baths and zinc ion crosslinking help improve the mechanical properties of the fibers and sutures. Immersing the sutures in a sericin solution also significantly improves their mechanical properties. Adding procaine further improves the mechanical properties of the sutures. The mechanical properties of sutures with different strand counts show that increasing strand count increases the mechanical properties of the sutures.

[0161] Figure 4 A and 4B show the effect of twist on the mechanical properties of sutures. Figure 4 As can be seen from Table 5, with the increase of twist, STZS 12 The mechanical properties of STZS show a trend of first increasing and then decreasing. Specifically, when the twist increases from 0 to 8 Twists / cm, 12 The mechanical strength of the STZS12 suture gradually increased, indicating that moderate twisting can effectively improve the mechanical properties of the fiber. However, when the twist is further increased to 12 twists / cm, the mechanical strength decreases significantly due to excessive compression and strain on the fiber's internal structure caused by excessive twisting. Therefore, 8 twists / cm was determined to be the optimal twist for STZS12 suture, which can maximize its mechanical properties while maintaining the integrity of the fiber structure.

[0162] Table 4 compares PSTZS of the same diameter 12 、STZS 12 and commercial 5-0 The mechanical strength of PSTZS. 12 and STZS 12 The maximum bearing capacity of the commercial 5-0 The maximum bearing capacity is only 6.15N, which is about PSTZS 12 and STZS 12 This result shows that the PSTZS prepared by the present invention 12and STZS 12 The suture is significantly superior to commercial sutures in terms of mechanical properties, has higher tensile strength and breaking strength, and can better meet the strict requirements for the mechanical properties of sutures in medical applications.

[0163] Figure 5 Compared with STZS 12 、TZS 12 TS 12 The mechanical properties test results of STZS6, TZS6 and TS6 show that zinc ion-assisted crosslinking can enhance the mechanical properties of fibers and sutures, and photo-crosslinked methacryloyl sericin coating can increase the friction between fibers, thereby improving the overall mechanical properties of sutures.

[0164] Knot tensile strength is one of the key indicators of suture performance. Figure 6 As shown, PSTZS 12 and STZS 12 It shows significant advantages in terms of knot tensile strength. Specifically, PSTZS 12 and STZS 12 The knot tensile strength is close to or better than commercial 5-0 It shows that it can provide reliable ligation performance in clinical applications, effectively preventing sutures from loosening or breaking, thereby ensuring stable healing of surgical wounds.

[0165] TZS 12 、STZS 12 、PSTZS 12 Electron microscopy characterization:

[0166] TZS 12 、STZS 12 、PSTZS 12 Freeze-dry and observe under a scanning electron microscope. Figure 7 As shown, TZS 12 The surface can be seen after the traction of high-strength fiber surface oriented crystal arrangement structure, STZS 12 、PSTZS 12 Pores formed after freeze-drying of the methacryloyl sericin coating can be seen on the surface, which is conducive to the sustained release of drugs and antibacterial ions.

[0167] Fourier transform infrared spectroscopy

[0168] Weigh 2 mg of PSTZS 12 、STZS 12 、TZS 12 TS 12Each sample was mixed with 200 mg of potassium bromide and thoroughly ground. The mixture was then pressed into tablets at a pressure of 20 MPa using a tablet press. The samples were then scanned using a Fourier transform infrared spectrometer (FTIR) in attenuated total reflectance (ATR) mode with a scanning wavelength range of 500-4000 nm and a scanning frequency of 32 times / second. The peak spectrum data obtained from the scans was recorded.

[0169] Freeze-dried silk fibroin (SF(raw)), methacryloyl sericin hydrogel (SerMA), and procaine powder (Prilocaine) were subjected to FTIR scanning at a wavelength range of 500-4000 nm and a scanning frequency of 32 times / second, and the peak spectrum data obtained from the scanning was recorded.

[0170] Infrared spectroscopy showed the molecular structural characteristics of SerMA, natural regenerated silk fibroin and procaine (such as Figure 8 A) SerMA at 1630 cm -1 The wave number of natural regenerated silk protein is 3280cm -1 The hydroxyl vibration or stretching peak appears at the wave number 1630cm -1 、1510cm -1 and 1230cm -1 The wavenumbers at these locations reflect the β-folding conformational characteristics of amides I, II, and III, respectively. The characteristic peaks of procaine include the stretching vibration of the secondary amine NH (3270 cm -1 ), amide NH stretching vibration (3050cm -1 ), amide CN stretching vibration (2938cm -1 ), amide C=O stretching vibration (1680cm -1 The benzene ring structure of the compound shows CH stretching and bending vibration absorption peaks (1659cm -1 、1590cm -1 、1460cm -1 , 1120cm -1 ), the characteristic absorption peak of the ortho-substituted benzene ring is located at 750cm -1 . Figure 8 B recorded PSTZS 12 、STZS 12 、TZS 12 TS 12 Characteristic absorption peak of suture line. TS 12 The sample was at 1630 cm -1 、1510cm -1 and 1230cm -1 The absorption peaks shown at represent the β-sheet configurations of amide I, amide II, and amide III, respectively.12 With TZS 12 542cm in the sample -1 The absorption peak at PSTZS is attributed to the characteristic vibration of Zn-O bond. 12 The sample exhibited not only the characteristic absorption peaks of silk fibroin and Zn-O bonds, but also the characteristic absorption signal of procaine. The experimental results confirmed that zinc ions can form ionic covalent bonds with serine residues in silk fibroin, and that procaine was successfully loaded into the SerMA coating and fixed to the suture surface through cross-linking.

[0171] Drug release and biocompatibility test results:

[0172] L929 fibroblasts in STZS 12 and PSTZS 12 Cultured in extract, Figure 9 A shows the cell proliferation at 24 hours, 48 ​​hours and 72 hours. The results showed that the number of living cells increased significantly over time, and STZS 12 、PSTZS 12 No significant difference was observed between the two groups. CCK-8 assay further confirmed that STZS 12 and PSTZS 12 The cells maintained good vitality ( Figure 9 C), indicating that the two sutures are non-toxic to cells and have good biocompatibility.

[0173] In order to promote comfortable wound healing, the sutures are loaded with analgesic prilocaine and zinc ions. By calculating the drug loading, it was found that PSTZS 12 The loading amount of zinc ions in the suture was 6.25 mg / cm and the loading amount of prilocaine was 4.22 mg / cm. 12 The cells were placed in PBS solution and their absorbance (OD value, Figure 9 D) The results showed that the release rate of prilocaine was 35.84% on the first day, reaching 67% after three days, and then gradually slowing down. The release rate of zinc ions was 21% on the first day, reaching 75% after five days, and then also gradually slowing down. The faster release rate of prilocaine than zinc ions may be due to prilocaine being located in the outer layer of the fiber, while zinc ions are located in the inner layer. In addition, the lower release rate of both on the first day may be related to the low solubility of prilocaine and zinc ions in water.

[0174] 24-hour fibroblast migration assay ( Figure 9 B and E) show that STZS 12 and PSTZS 12It can significantly promote the migration of fibroblasts, which shows that these two sutures not only have good biocompatibility, but also can accelerate wound healing by promoting cell migration.

[0175] To evaluate STZS 12 and PSTZS 12 The biocompatibility of the sutures was investigated by subcutaneous implantation in rats, and the samples were observed 3, 7, 14, and 28 days after surgery (e.g. Figure 10 A). Figure 10 B The results showed that STZS 12 and PSTZS 12 Sutures triggered a mild inflammatory response after implantation, with a small amount of macrophage infiltration observed around the sutures 3 days after surgery. However, this inflammatory response gradually subsided over time, with a significant decrease at 14 and 28 days after surgery.

[0176] In vitro antimicrobial evaluation

[0177] Table 7 Diameter of Escherichia coli inhibition zone of different sutures (mm)

[0178]

[0179]

[0180] Table 8 Diameter of Staphylococcus aureus inhibition zone of different sutures (mm)

[0181]

[0182] Tables 7 and 8 show the diameters of the inhibition zones of different sutures against Escherichia coli and Staphylococcus aureus, respectively. An inhibition zone diameter greater than 10 mm indicates that the sample has a significant antibacterial effect. Figure 11 As shown in A, 5-0 Suture, TS 12 and STS 12 The extracts of TZS showed no obvious inhibition zone on the E. coli (ATCC 25922) culture plate, indicating that silk fibroin and SerMA had no obvious antibacterial effect. 12 、STZS 12 and PSTZS 12 The extract showed significant antibacterial effect on these bacterial plates.

[0183] like Figure 11 A and Table 7, on E. coli plates, TZS 12 、STZS 12 and PSTZS 12On the first day, the diameters of the inhibition zones were 11.98 mm, 13.51 mm, and 14.03 mm, respectively; on the second day, the diameters of the inhibition zones were approximately 2.77 mm, 7.24 mm, and 7.05 mm, respectively; and on the third day, the diameters of the inhibition zones further shrank to 1.13 mm, 2.31 mm, and 2.54 mm. This indicates that the size of the inhibition zones gradually decreases with time and with decreasing zinc ion concentrations. Figure 11 B, STZS 12 and PSTZS 12 The release of zinc ions shows a certain sustained release characteristic, thus having a sustained antibacterial ability.

[0184] Figure 11 The results of scanning electron microscopy (SEM) observation of C further confirmed this: E. coli was able to colonize and adhere to 5-0 Suture, TS 12 and STS 12 The surface of TZS 12 、STZS 12 and PSTZS 12 No bacterial colonization was observed on the surface of STZS. 12 and PSTZS 12 It has a sustained antibacterial effect on Escherichia coli and can effectively inhibit the adhesion and growth of bacteria.

[0185] exist Figure 12 A and Table 8, 5-0 Suture, TS 12 and STS 12 The extracts of TZS showed no significant inhibition zone on the culture plate of Staphylococcus aureus (ATCC25923), and the surface silk fibroin and SerMA had no obvious antibacterial effect. 12 、STZS 12 and PSTZS 12 The extracts of TZS showed significant antibacterial effects on these bacterial plates. 12 、STZS 12 and PSTZS 12 The diameters of the inhibition zones on the Staphylococcus aureus plates were 12.16 mm, 14.91 mm, and 16.08 mm on the first day, respectively; on the second day, they shrank to 6.20 mm, 9.01 mm, and 9.78 mm, respectively; and on the third day, the diameters further decreased to 0 mm, 5.06 mm, and 5.33 mm. This indicates that the size of the inhibition zone gradually decreases with decreasing zinc ion concentration.

[0186] like Figure 12 B, STZS 12 and PSTZS 12The release of zinc ions shows a certain sustained release characteristic, thus having a sustained antibacterial ability. Figure 12 The results of scanning electron microscopy (SEM) observation of C further confirmed this: Staphylococcus aureus was able to colonize and adhere to 5-0 Suture, TS 12 and STS 12 The surface of TZS 12 、STZS 12 and PSTZS 12 These results indicate that STZS 12 and PSTZS 12 It has a sustained antibacterial effect on Staphylococcus aureus and can effectively inhibit the adhesion and growth of bacteria.

[0187] Figure 13 B The results showed that STZS 12 and PSTZS 12 The wound closure speed of the group was faster and the inflammatory response was also better than that of the 5-0 In the first three days after surgery, all groups developed a scab, a common early sign of healing. By day 5, STZS 12 and PSTZS 12 The wounds of the 5-0 The group still had obvious crusts and inflammation. This may be due to the 5-0 The high tension of the sutures may cause tissue damage, bleeding, and inflammation. The STZS group still had some inflammation, while 12 and PSTZS 12 By day 14, the healing process was complete in all groups.

[0188] Histological analysis by hematoxylin-eosin (H&E) staining revealed Figure 13 C), On days 1 and 3, scabs began to cover the edges of the incision (green arrows indicate the direction of the edges). By day 5, 5-0 The STZS group had extensive scab formation due to tissue damage caused by high-tension sutures, while the 12 and PSTZS 12 The wounds of the PSTZS group began to close. 12 The closure speed is faster. By the 7th day, 5-0 The epidermis of the group healed with a scab, but the dermis still had an open incision, while the STZS 12 and PSTZS 12 The wounds of the 5-0 group were completely closed. The incision edges of the STZS group had closed, and many inflammatory cells assisted in the repair. 12 and PSTZS 12 The skin of the group became smoother, while the 5-0 The group still has a noticeable depression.

[0189] To verify the STZS 12 and PSTZS 12 The organ toxicity of these materials was examined in rats' hearts, livers, spleens, lungs, and kidneys 28 days after subcutaneous implantation of these materials. Figure 13 No obvious toxic effects were observed in these organs, which maintained normal structure and function, similar to those of normal rats.

[0190] In summary, the present invention has successfully developed an innovative biomimetic silk protein multifunctional hydrogel suture. This suture not only exhibits excellent mechanical properties, but also has good cell compatibility and excellent in vitro and in vivo biocompatibility. Through the introduction of light-cured SerMA coating, the suture is endowed with a sustained drug release function, thereby achieving long-term sustained release of drugs. In in vitro antibacterial experiments, the suture can significantly inhibit the growth of Staphylococcus aureus and Escherichia coli, showing strong antibacterial properties. In addition, the results of in vitro and in vivo experiments show that STZS 12 and PSTZS 12 The two sutures can effectively promote wound healing and accelerate the tissue repair process. This invention proposes a simple and promising preparation strategy, opening up a new path for the development of high-strength, long-lasting, and multifunctional new sutures, which are expected to be widely used in clinical medicine.

[0191] The embodiments provided above are not intended to limit the scope of the present invention, nor are the steps described to limit their execution order. Any obvious improvements to the present invention made by those skilled in the art in combination with existing common knowledge shall fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a biomimetic regenerated silk protein multifunctional hydrogel suture, characterized in that: The following steps are involved: (1) Extraction of regenerated silk fibroin: natural silk cocoons were washed, dried, degummed, dried, dissolved, dialyzed in a 3500 Da dialysis bag, filtered, centrifuged, and freeze-dried to obtain silk fibroin; (2) preparing regenerated silk fibroin hydrogel fibers by wet spinning: dissolving the silk fibroin obtained in step (1) in formic acid to prepare a spinning solution, pumping the spinning solution into a methanol coagulation bath through a hose using a microfluidic pump, and orienting and pulling the prepared fibers using a multifunctional mechanical tester. The pulled fibers are immersed in a recrystallization bath for recrystallization and solidified and dried to obtain high-strength silk fibroin hydrogel fibers; (3) Antibacterial treatment: soaking the high-strength silk fibroin hydrogel fiber obtained in step (2) in a 0.1-5% zinc chloride aqueous solution for 4-6 hours, taking out the fiber and sterilizing it to obtain a high-strength, antibacterial regenerated silk fibroin hydrogel fiber; (4) Preparation of suture: twisting the high-strength, antibacterial regenerated silk fibroin hydrogel fibers obtained in step (3) into suture; (5) Preparing a light-cured methacryloyl sericin solution: dissolving sericin powder in a phosphate buffer solution, adding methacrylic anhydride in a dark environment, reacting for 12 to 72 hours to obtain a methacryloyl-modified sericin solution, dialyzing, and freeze-drying to obtain the methacryloyl-modified sericin; preparing a methacryloyl-modified sericin solution with a mass concentration of 5%, mixing the methacryloyl-modified sericin solution with a photoinitiator solution in a dark environment to obtain a light-cured methacryloyl sericin solution; (6) Assembly of biomimetic silk protein multifunctional hydrogel suture: The suture was immersed in a procaine-loaded photocurable methacryloyl sericin solution for 2-3 minutes, and the suture was taken out and sterilized to obtain a biomimetic silk protein multifunctional hydrogel suture.

2. The preparation method according to claim 1, characterized in that In step (1), the degumming process is as follows: taking the washed and dried silk cocoons and placing them in a Na2CO3 solution, boiling them for 1 hour to degumming, the mass volume ratio of the silk cocoons to the Na2CO3 solution is 25-35g / 10-14L, and the concentration of the Na2CO3 solution is 0.02-0.025mol / L. The dissolution process is specifically as follows: taking the degummed and dried silk cocoons and dissolving them in a lithium bromide solution with a concentration of 9-9.4mol / L, stirring and dissolving them in a water bath at 60°C for 4-6 hours, wherein the mass volume ratio of the degummed and dried silk cocoons to the lithium bromide solution is 25-27g:90-100mL.

3. The preparation method according to claim 1, characterized in that In step (2), the concentration of the spinning solution is 12-15%, the speed of pumping the spinning solution into the methanol coagulation bath is 0.5-1 mL / min, the diameter of the hose is 200 μm, and the spinning solution is immersed in the 95% methanol coagulation bath for 10-30 min. The methanol coagulation bath also contains zinc chloride, and the mass concentration of the zinc chloride is 0.5-5%.

4. The preparation method according to claim 1, characterized in that In step (2), the ambient temperature during the orientation traction is 23-25°C, the air humidity is 50%, the traction speed is v=200mm / min, and after the orientation traction, the tension is fixed with a force of 1N for 2-3 minutes. The traction multiple of the orientation traction is 4-8 times.

5. The preparation method according to claim 1, characterized in that In step (2), the solution of the recrystallization bath is a 75% ethanol solution, the fiber is in the recrystallization bath for 10 minutes, the curing temperature is 25-37° C., the curing time is 10-30 minutes, and the solution of the recrystallization bath further contains zinc chloride, and the mass concentration of the zinc chloride is 0.5-5%.

6. The preparation method according to claim 1, characterized in that In step (4), the specific process of twisting into suture thread is to twist the high-strength, antibacterial regenerated silk fibroin hydrogel fiber in a Z-shape with a twist of 1 to 12 twists / cm, and then dry and fix it.

7. The preparation method according to claim 1, characterized in that In step (5), the mass-to-volume ratio of sericin powder to phosphate buffer is 0.8-1 g:4-5 mL, the mass ratio of methacrylic anhydride to sericin is 3:5, and dialysis is performed in a dialysis bag with a molecular weight cutoff of 3500 Da. The dialysis time is 48-72 hours, and the water is changed 2-3 times per day. The photoinitiator is Irgacure 2959, the concentration of the photoinitiator solution is 0.01-0.02 g / mL, and the volume ratio of the methacrylated sericin solution to the photoinitiator solution is 100-1000:

1.

8. The preparation method according to claim 1, characterized in that In step (6), the preparation method of the procaine-loaded photocurable methacryloyl sericin solution is to disperse procaine in the photocurable methacryloyl sericin solution, wherein the mass volume ratio of procaine to methacryloyl sericin solution is 2.5-5 g:100 mL. 9 . The biomimetic regenerated silk protein multifunctional hydrogel suture thread prepared according to the preparation method of any one of claims 1 to 8 .

10. Application of the biomimetic regenerated silk protein multifunctional hydrogel suture according to claim 9 in the fields of biomedicine and tissue engineering.

Images