Preparation method of a controllable degradation silk medical suture

Abstract

The present invention discloses a preparation method of a controllable degradation silk medical suture, comprising the steps of: braiding a silk shell thread and a core thread on a braiding machine to obtain a braided suture; performing degumming treatment to obtain a degummed suture; preparing a serine protease solution; soaking the degummed suture in the serine protease solution for enzyme pretreatment; performing a coating treatment on the suture after enzyme pretreatment with a functional silk fibroin solution to obtain a coated suture; performing fumigation and heat setting treatment on the coated suture to obtain a controllable degradation silk medical suture. This suture has good mechanical strength, reduces the crystallinity of silk, and meets the requirements of absorbable sutures. At the same time, it retains the advantages of silk such as degradability and excellent biocompatibility. The functional coating endows silk with good antibacterial and degradation-promoting properties, compensating for the rapid loss of mechanical properties. The degradation rate and mechanical properties of the suture are controllable, and the method is simple, easy to operate and feasible.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and relates to a preparation method of a controllable degradation silk medical suture. Background Art

[0002] Sutures are classified into absorbable sutures and non-absorbable sutures according to the absorbability of the materials. Absorbable sutures avoid the trouble caused by suture removal, and their degradation products can be metabolized and excreted by the human body. Existing absorbable sutures usually use synthetic materials, mostly polymerized from several monomers. The monomer fragments generated by degradation may cause swelling and local inflammation at the implantation site. The support time of synthetic absorbable sutures is often short, and the tension is small. It is difficult to meet the requirements in some parts that require large tension, and there is a risk of fracture. Synthetic sutures usually have no promoting effect on wound healing and repair. The degradation products of the vast majority of synthetic materials are single and may be acidic, resulting in certain biocompatibility problems.

[0003] Silk is composed of natural materials, fibroin and sericin. Sericin is located on the outer layer of silk fiber and mainly plays a bonding role. After removing sericin protein from silk, it becomes fibroin protein. Fibroin protein constitutes the core fiber of silk and plays a key role in mechanical properties. Silk sutures have been used as commercial surgical sutures for decades. A large number of hospital clinical results have proved that degummed silk sutures will not have an adverse effect on the human body and will not cause an inflammatory reaction after being implanted into human tissues. The main component of fibroin protein is amino acids. Among them, glycine, alanine, and serine are the main amino acids that make up the crystalline region, accounting for about 85% of the total amino acid molar percentage. Serine, aspartic acid, and tyrosine with hydrophilic groups account for about 30% of the total amount. As a natural protein, fibroin protein can promote wound healing. Compared with other synthetic material sutures, the probability of scarring is smaller, and there is great market potential.

[0004] After being implanted in the body for two months, the silk suture retains more than 50% of its mechanical properties. Silk is biodegradable and its degradation products are amino acids. However, due to the presence of crystalline regions rich in glycine and alanine in fibroin, it has a relatively strong resistance to protease degradation, so the degradation time is relatively long. In order to accelerate the degradation of silk sutures, researchers mainly regulate it through methods such as wet spinning, degumming, coating, and genetic engineering to explore silk sutures with accelerated degradation. For example, a kind of absorbable suture that uses sodium carbonate, sodium bicarbonate, and calcium chloride to degum braided silk sutures and then coats them with a fibroin solution (CN109385710A). However, the suture prepared by this method only uses the degumming method to remove the sericin on the surface of silk, and then coats it with a fibroin solution, which cannot specifically damage the crystalline region of fibroin. The fibroin coating will delay degradation and cannot achieve the effect of accelerated degradation and absorption. Another example is to obtain a braided scaffold by coating a collagen solution after silk degumming (CN109758262A). This method also does not damage the crystalline region of fibroin, and the coated collagen cannot promote degradation. Another example is a biodegradable absorbable suture prepared by using sodium alginate and casein to prepare a spinning solution through wet spinning and other methods (CN115192764A). However, the artificial regenerated silk obtained by wet spinning has an unstable structure, its mechanical properties are inferior to natural silk, and the suture composition is single and the components are not easy to control. Another example is through genetic engineering methods. Using the heavy chain recombinant protein gene of silkworm and protease K, α-I collagenase cleavage sequence recognition design genes, then expressing fibroin through biological fermentation methods, and then preparing regenerated sutures through wet spinning (CN112301495A and CN112301496A). On the one hand, the regenerated silk obtained by wet spinning has poor mechanical strength, and its toughness and ductility are inferior to silk. On the other hand, the gene design is difficult, the cost is high, and the practical operation feasibility is not high. In the above methods, there are often problems such as not damaging the crystalline region of silk, the structure and mechanical properties of regenerated fibroin being unsatisfactory, and the biodegradability and biocompatibility of materials not being comparable to those of the original silk, etc., and it is necessary to further improve the preparation methods and processes. In order to overcome the defects of the existing technology and solve the above problems, it is necessary to develop a preparation method for a new type of controllable degradation silk medical suture.

[0005] Therefore, in order to solve the problems of slow degradation of existing silk sutures, low mechanical strength, single component, and difficult to control the degradation rate of sutures obtained by methods such as regenerated silk, it is necessary to prepare a controllable degradation silk medical suture. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method for a controllable degradation silk medical suture to solve the above problems.

[0007] The technical solution of the present invention is:

[0008] A preparation method of a controllable degradation silk medical suture, specifically including:

[0009] (1) Weave silk shell thread and core thread on a knitting machine to obtain a knitted suture;

[0010] (2) Degum the knitted suture to obtain a degummed suture;

[0011] (3) Prepare a serine protease solution;

[0012] (4) Immerse the degummed suture in the serine protease solution for enzyme pretreatment;

[0013] (5) Coating treatment of the suture after enzyme pretreatment with a functional silk fibroin solution to obtain a coated suture;

[0014] (6) Fumigation and heat setting treatment of the coated suture to obtain a controllable degradation silk medical suture.

[0015] Further, in step (3), the preparation of the serine protease solution is specifically: dissolve serine protease in a buffer solution, and configure it into a 0.1 - 1000U / mL serine protease solution according to the titer and activity at 0.01 - 10mg / mL, gently shake, avoid light and violent stirring, and carry out aging treatment in a hot air stream at a temperature of 20 - 80°C for 1 - 10min for further dissolution, and store it in the dark at -20 or -80°C to obtain the serine protease solution.

[0016] Further, the serine protease is any one of α-chymotrypsin, proteinase K, proteinase XXI, and proteinase XIV.

[0017] Further, the buffer solution is any one of 0.01 - 1M phosphate buffer solution, phosphate buffer solution, deionized water, Tris-HCl buffer solution, acetic acid-sodium acetate buffer solution, and sodium citrate buffer solution. The pH value range of the buffer solution is 5 - 9. The buffer solution is pre-sterilized by high temperature and high pressure, and the sterilization temperature is 120 - 140°C, and the time is 20 - 40min.

[0018] Further, before step (4), first immerse the degummed suture in a phosphate buffer solution, ultrasonically clean it for 5 - 30min, and then rinse it with deionized water and dry it.

[0019] Further, in step (4), the serine protease solution is selected from any two of α-chymotrypsin solution, proteinase K solution, proteinase XXI solution, and proteinase XIV solution, and they are mixed and synergistically treated in a ratio of 1:1 - 10 or 1 - 10:1. Alternatively, the serine protease solution is selected as proteinase K solution, or after soaking in one of the serine protease solutions among α-chymotrypsin, proteinase K, proteinase XXI, and proteinase XIV, it is then soaked in another serine protease solution among α-chymotrypsin, proteinase K, proteinase XXI, and proteinase XIV for 1 - 72 h. The bath ratio is 1:100 - 1000, and the bath ratio of the degummed suture to the serine protease solution is 1:100 - 1000. The temperature of the enzyme pretreatment is 25 - 90 °C, and the time is 1 - 72 h.

[0020] Further, after step (4), the degummed suture is soaked in a phosphate buffer solution and treated at a temperature of 100 - 120 °C for 25 - 20 min, or boiled in water bath for 30 - 60 min to inactivate the protease, and then washed thoroughly with water to remove the remaining enzyme solution on the surface and air-dried overnight.

[0021] Further, in step (5), the method for preparing the functional silk fibroin solution is as follows: any one or more of serine protease, fluorescent substance, antibacterial drug, antibacterial particle, or interfacial connection substance are added to a silk fibroin solution with a mass percentage of 0.1 - 10%. The mass percentage of any one or more of the serine protease, fluorescent substance, antibacterial drug, antibacterial particle, or interfacial connection substance to the silk fibroin solution is 1:10 - 1000. After mixing evenly, a functional silk fibroin solution is obtained. Among them, the serine protease is any one of α-chymotrypsin, proteinase K, proteinase XXI, and proteinase XIV; the fluorescent substance is rhodamine B or calcein; the antibacterial drug is any one of berberine, curcumin, and penicillin; the antibacterial particle is any one of nano-zinc oxide, nano-silica, and graphene; the interfacial connection substance is any one of chitosan, sodium alginate, and sodium hyaluronate.

[0022] Further, in step (5), the specific process of coating the enzyme-pretreated suture with the functional silk fibroin solution to obtain a coated suture is as follows: Immerse the enzyme-pretreated suture in the functional silk fibroin solution with a bath ratio of 1:100 - 1000, soak it at a temperature of 25 - 90 °C for 20 - 60 min, then take it out, rinse it with deionized water, dry it at a temperature of 100 - 130 °C for 1 - 3 min, and then dry it at a temperature of 40 - 80 °C for 1 - 3 h to obtain a silk fibroin-coated suture with a relatively high content of β-sheet structure; or naturally air-dry the soaked suture at a temperature of 0 - 40 °C to obtain a silk fibroin-coated suture mainly composed of random coils and α-helix structures; or freeze the soaked suture at a temperature of -20 °C or -80 °C for 1 - 6 h and then lyophilize it to obtain a silk fibroin-coated suture mainly composed of the easily absorbable Silk I crystal form.

[0023] Further, in step (6), the specific process of fumigating and heat-setting the coated suture to obtain a controllable degradation silk medical suture is as follows: Fumigate and heat-set the coated suture in any one of methanol vapor, ethanol vapor, and water vapor at a temperature of 25 - 120 °C for 1 - 72 h to obtain a controllable degradation silk medical suture.

[0024] The present invention provides a method for preparing a controllable degradation silk medical suture, and its advantages are as follows:

[0025] (1) The present invention uses a serine protease solution, which can act specifically on the main amino acids such as glycine, alanine, and serine in the crystalline region sequence of silk, regulate the size and orientation structure of the crystalline region, and promote degradation. The enzymatic reaction has strong specificity, high efficiency, and mild reaction conditions. The degradation degree can be regulated by controlling the concentration, temperature, pH, and time. The process is simple, the operation is easy, and the protease can be removed through soaking, rinsing, etc.

[0026] (2) The present invention adopts a silk composite braiding and forming technology. By selecting positive and negative twist yarns as braiding materials, and using shell yarns and core yarns with different linear densities for composite braiding, a relatively thin yarn is selected as the shell yarn for braiding, and a relatively thick yarn is used as the core for support, and shell-core composite braiding and forming are carried out to further improve the mechanical properties; the wire diameter and knot-breaking strength of the prepared controllable degradation silk medical suture meet the requirements of the absorbable suture standard YY 1116 - 2020.

[0027] (3) The present invention uses a water-soluble silk fibroin coating, fumigation, freeze-drying, and heat setting treatments to obtain a silk fibroin coating with an α-helix, random coil, or β-sheet structure. The coating can fill the cracks and defects treated by silk enzymes to make up for the insufficient mechanical properties. The degradation rate of the coating obtained by different treatment methods is controllable and the degradation rate is faster than that of the original silk. Methanol, ethanol, and steam fumigation are used to promote the formation of β-sheets in the water-soluble protein, further making up for the loss of mechanical properties caused by the enzymatic degradation treatment. Silk fibroin is naturally green and environmentally friendly, non-toxic, harmless, safe, and has excellent performance. Adding functional substances can further accelerate degradation, endow antibacterial functions, and improve mechanical properties, showing good application prospects.

[0028] (4) The method of the present invention is efficient and practical. The controllable degradation silk fibers obtained by enzymatic degradation can be applied to other woven biomedical textiles, such as artificial blood vessels, heart valves, artificial tendons, artificial ligaments, nerve conduits, etc. woven from controllable degradation silk, which is a new type of controllable degradation method. Description of the Drawings

[0029] Figure 1 Scanning electron microscope morphology diagram (x50) of the silk suture promoted by protease K in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0030] Figure 2 Scanning electron microscope morphology diagram (x1000) of the silk suture promoted by protease K in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0031] Figure 3 Scanning electron microscope morphology diagram (x5000) of the silk suture promoted by protease K in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0032] Figure 4 Graph of the thread diameter and mass change in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0033] Figure 5 Graph of the change in the knot tensile breaking strength in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0034] Figure 6 Infrared spectrum diagram and fitting result diagram in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0035] Figure 7 X-ray diffraction diagram and fitting result diagram in Example 1 of the preparation method of a controllable degradation silk medical suture according to the present invention;

[0036] Figure 8 Differential scanning calorimetry chart and fitting result chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0037] Figure 9 Amino acid analysis result chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0038] Figure 10 Raman spectrogram of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0039] Figure 11 In vitro simulated degradation scanning electron micrograph of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0040] Figure 12 In vitro simulated degradation infrared spectrum fitting result chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0041] Figure 13 In vitro simulated degradation knotting tensile strength change chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0042] Figure 14 In vitro simulated degradation X-ray diffraction chart and fitting result chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0043] Figure 15 In vitro simulated degradation differential scanning calorimetry chart and fitting result chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0044] Figure 16 In vitro simulated degradation Raman spectrogram of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0045] Figure 17 Coating and in vitro simulated degradation scanning electron micrograph of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0046] Figure 18 Coating and in vitro simulated degradation knotting tensile fracture strength change chart of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0047] Figure 19 Drug loading and antibacterial effect chart of the coating of the preparation method of a controllable degradation silk medical suture according to the present invention in Example 1;

[0048] Figure 20 Distribution map of the fluorescent substance of the coating in Example 4 for the preparation method of a controllable degradation silk medical suture according to the present invention. Detailed implementation manners

[0049] The present invention draws on the idea of twisting the "steel cables" of a suspension bridge to improve the mechanical properties of silk sutures. The yarns with a positive and negative twist structure will stretch and contract with the change of stress, meeting the requirements of passing through the skin and tissues. The original silk is etched by enzyme pretreatment, retaining the crystal structure and some properties of the original silk while partially destroying the crystalline region targeted, promoting the hydrolysis of the silk suture. The biocompatibility and biodegradability of the material are superior to those of synthetic materials. On the basis of the enzyme-pretreated silk suture as the "steel cable", a soluble silk fibroin solution is used as a functional coating to fill the pores and cracks formed by the enzyme pretreatment. The mechanical properties are improved through coating, fumigation, freeze-drying, and heat setting, and antibacterial particles, antibacterial drugs, fluorescent substances, and serine protease are added to the coating to modify the silk suture and regulate the mechanical properties and degradation rate, achieving the effect of "broken but not disconnected". The medical silk suture obtained by this method promotes degradation and maintains good mechanical properties, and can be extended to application scenarios such as braided degradable artificial tendon / ligament scaffolds, heart valves, and nerve conduits.

[0050] In some embodiments of the present invention, silk yarns are used as raw materials, and multiple silk shell and core yarns with different linear densities are used to braid sutures. Serine protease, which hydrolyzes hydrophobic amino acids relatively fast, is used to degrade silk fibroin, reducing the content of the crystalline region structure in silk and accelerating degradation. A silk fibroin solution with a concentration of 0.1-10% by mass is selected for coating. If the concentration is too low, the mechanical improvement is not significant; if the concentration is too high, it is easy to form a gel. Protease is added to the coating solution to improve the degradability after coating, antibacterial particles and antibacterial drugs are added to endow antibacterial functions, and interface connection substances and silk fibroin coating are used to form hydrogen bond crosslinking to further improve the coating binding strength and enhance the mechanical properties.

[0051] The specific operation steps of the preparation method of the controllable degradation silk medical suture are as follows:

[0052] S1, braid the silk shell and core yarns on a braiding machine to obtain a braided silk suture.

[0053] Use 2 1-3 ply 1-10 tex silk shell yarns with a twist of 400-1200Z+500-900S in both positive and negative directions and 1-3 ply 4-60 tex silk core yarns to braid a braided silk suture on an 8-64 spindle braiding machine.

[0054] S2. Degum the braided silk suture, then wash and dry it to obtain a degummed suture.

[0055] Immerse the suture in a 0.01 - 0.06M or 0.01 - 0.1% (w / v) sodium carbonate or sodium bicarbonate solution at 50 - 100°C or a 1 - 100U / mL papain, trypsin, or streptomyces protease solution at 30 - 80°C for 10 - 60 minutes. The bath ratio of the suture to the degumming solution is 1:10 - 1000. Rinse it with deionized water 1 - 3 times, and then dry it to obtain a degummed suture.

[0056] S3. Prepare a serine protease solution.

[0057] Dissolve α - chymotrypsin (from bovine pancreas, porcine pancreas), proteinase K (from Candida albicans), proteinase XXI (from Streptomyces griseus), and proteinase XIV (from Streptomyces griseus) in a 0.01 - 1M, pH = 5 - 9 phosphate buffer, phosphate buffer, deionized water, Tris - HCl buffer, acetic acid - sodium acetate buffer, or sodium citrate buffer that has been autoclaved at 120 - 140°C for 20 - 40 minutes. Prepare a 0.1 - 1000U / mL serine protease solution at 0.01 - 10mg / mL according to the titer and activity. Gently shake it, avoid light and vigorous stirring, and age it in a hot air stream at 20 - 80°C for 1 - 10 minutes for further dissolution. Store it in the dark at - 20 or - 80°C to obtain a serine protease solution. Avoid repeated freezing and thawing when using it.

[0058] S4. Immerse the degummed suture in the serine protease solution for enzyme pretreatment.

[0059] The degummed suture is soaked in phosphate buffer and ultrasonically cleaned for 5 - 30 min, then rinsed with deionized water and dried. It is soaked in a solution of two different serine proteases selected from 0.1 - 1000 U / mL α-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (Candida albicans), proteinase XXI (Streptomyces griseus), and proteinase XIV (Streptomyces griseus) in a ratio of 1:1 - 10 or 1 - 10:1, or soaked in a solution of one of the serine proteases selected from α-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (Candida albicans), proteinase XXI (Streptomyces griseus), and proteinase XIV (Streptomyces griseus) for 1 - 72 h and then soaked in another solution of one of these serine proteases for 1 - 72 h. The bath ratio is 1:100 - 1000. Since heat flow is required during enzyme pretreatment, it is placed in a forced-air oven or drying oven with heat flow. Subsequently, the silk suture is soaked in phosphate buffer, treated at 100 - 120 °C for 5 - 20 min or boiled in water bath for 30 - 60 min to inactivate the protease, then thoroughly washed with water to remove the remaining enzyme solution on the surface, and air-dried overnight.

[0060] S5. The suture after enzyme pretreatment is impregnated, coated, and dried with a functional silk fibroin solution to obtain a coated suture.

[0061] Add one of serine proteases α-chymotrypsin (from bovine pancreas, porcine pancreas), proteinase K (from Candida albicans), proteinase XXI (from Streptomyces griseus), proteinase XIV (from Streptomyces griseus), or one of fluorescent substances rhodamine B, calcein, or one of antibacterial drugs such as berberine, curcumin, penicillin, or one of antibacterial particles nano-zinc oxide, nano-silica, graphene, or one of interfacial connection substances chitosan, sodium alginate, sodium hyaluronate into a silk fibroin solution with a mass percentage of 0.1 - 10%. The ratio of the above substances to the silk fibroin solution (mass percentage) is 1:10 - 1000. Stir evenly to obtain a functional silk fibroin solution. Immerse the suture pretreated with enzyme into the functional silk fibroin solution with a bath ratio of 1:100 - 1000, soak at 25 - 90 °C for 20 - 60 min, take out and gently rinse with deionized water. Dry at 100 - 130 °C for 1 - 3 min to improve the binding fastness, and then dry at 40 - 80 °C for 1 - 12 h to obtain a silk fibroin-coated suture with a relatively high content of β-sheet structure. Or take out the soaked coated suture and place it in a fume hood at 0 - 40 °C to air dry naturally to obtain a silk fibroin-coated suture mainly composed of random coils and α-helix structures. Or take out the coated suture, dip-roll it, and then freeze it at -20 °C or -80 °C for 1 - 6 h, and then lyophilize it to obtain a silk fibroin-coated suture mainly composed of the easily absorbable Silk I crystal form.

[0062] S6. Fumigate and thermally set the coated suture to obtain a controllable degradation silk suture.

[0063] Fumigate and thermally set the coated suture at 25 - 120 °C in methanol vapor or ethanol vapor or water vapor for 1 - 72 h to promote the formation of β-sheets from the random coils and α-helices of the water-soluble silk fibroin in the coating to make up for the mechanical loss, and obtain a controllable degradation silk medical suture.

[0064] To make the above objects, features, and advantages of the present invention more obvious and understandable, the technical solutions of the present invention will be further described below with specific embodiments. However, the present invention is not limited to the listed embodiments, and should also include any other well-known changes within the scope of the rights required by the present invention.

[0065] As used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that mutually excludes other embodiments. Example 1

[0066] This example demonstrates a preparation method of a controllable degradation silk medical suture, and the specific steps are as follows:

[0067] (1) Weaving of silk suture: Fibers with different core-sheath yarn ratios are woven using an 8-64 spindle loom. Two-ply silk with a twist of 400-1200Z + 500-900S in both forward and reverse directions is used for weaving. Silk with a tex of 1-10 is used as the sheath yarn, and silk with a tex of 5-60 is used as the core yarn. According to the requirements in the YY 1116-2020 standard, braided medical sutures of sizes 12-0, 11-0, 10-0, 9-0, 8-0, 7-0, 6-0, 5-0, 4-0, 4-0 / T, 3-0, 2-0 / T, 2-0, 1-0, 1, 2, 3, 4, and 5 are designed, with a wire diameter of 0.001-0.950 mm.

[0068] (2) Degumming of silk suture: The silk suture is immersed in a sodium carbonate solution with a concentration of 0.01-1 g / L, treated at a slightly boiling state for 30-60 min, then rinsed with deionized water multiple times and air-dried overnight in a fume hood.

[0069] (3) Preparation of serine protease solution: Serine protease K and protease XIV are mixed at a ratio of 1:1-10 or 1-10:1, dissolved in deionized water, phosphate buffer, or Tris-HCl solution with a pH of 5-9, with a concentration of 1-300 U / mL, and placed at 10-70 °C for 1-30 min.

[0070] (4) Enzyme pretreatment of silk suture: The degummed suture is soaked in a mixed solution of protease K and protease XIV, with a bath ratio of 1:10-1000, and treated in a forced-air drying oven at 10-100 °C for 1-48 h.

[0071] (5) Coating with functional silk fibroin solution: Berberine, curcumin, and penicillin with a mass percentage of 0.1-1% are added to the silk fibroin solution to obtain a drug-loaded silk fibroin solution, or protease XIV, protease XXI, protein K, and α-chymotrypsin are added to obtain a silk fibroin solution carrying serine protease. The enzyme-pretreated silk suture is immersed in the drug-loaded or protease-carrying silk fibroin solution at 40-80 °C for 20-60 min, then dried at 100-130 °C for 1-3 min, then dried at 40-60 °C, pre-frozen at -80 °C for 1-6 h, and then freeze-dried to obtain a coated suture.

[0072] (6) Fumigation of coated suture: The dried suture is placed in methanol vapor, ethanol vapor, or water vapor and fumigated at 40-100 °C for 3-24 h to obtain a controllable degradable silk suture. Example 2

[0073] This example demonstrates a method for preparing a controllable degradable silk medical suture, and the specific steps are as follows:

[0074] (1) Degumming of silk yarn: The shell yarn of the suture is made of two strands of 1-10 tex silk yarn, and the core yarn is made of two strands of 5-60 tex silk yarn. Add 10-20 L of distilled water to a water bath, bring to a boil, add 0.1-10 g of sodium carbonate with a concentration of 0.01-1 mol / L, immerse the silk in the solution, boil for 30-60 min, then rinse in distilled water and dry overnight at room temperature.

[0075] (2) Weaving of silk suture: Weave the degummed silk yarn on an 8-64 spindle loom to obtain a suture with a wire diameter of 0.001-0.950 mm and mechanical properties meeting the requirements of standard YY 1116-2020.

[0076] (3) Preparation of serine protease solution: Mix streptococcal protease XIV or XXI and proteinase K at a ratio of 1:1-10 or 1-10:1 and dissolve in deionized water, PBS solution or Tris-HCl solution with pH = 5-9. The enzyme dosage is 1-100 mg / L to prepare a solution with a concentration of 1-300 U / mL, and age at a temperature of 30-50 °C for 1-30 min.

[0077] (4) Enzyme pretreatment of silk suture: Immerse the degummed silk suture in a mixed solution of streptococcal protease and proteinase K with a bath ratio of 1:10-1000, place it in an oven at 10-100 °C, and pass a nitrogen or carbon dioxide gas stream around the sample at a flow rate of 200-1000 mL / min for 1-48 h. Gently shake every 30-60 min to ensure full and uniform contact between the silk suture and the protease solution.

[0078] (5) Coating with functional silk fibroin solution: Add streptococcal protease with a mass percentage of 0.1-1% to the silk fibroin solution to obtain a silk fibroin solution for promoting degradation. Immerse the streptococcal protease-pretreated silk suture in the silk fibroin solution for promoting degradation at 20-30 °C for 20-60 min, then gently rinse with distilled water, and repeat the soaking and rinsing 1-20 times. Then pre-freeze at -20 °C or -80 °C for 1-6 h and then freeze-dry to obtain a coated suture.

[0079] (6) Fumigation of coated suture: Place the dried suture in steam for fumigation treatment at 100-120 °C for 3-72 h, and then vacuum dry to obtain a controllable degradation silk suture. Example 3

[0080] This example demonstrates a method for preparing a controllable degradation silk medical suture, and the specific steps are as follows:

[0081] (1) Weaving of silk suture: Fibers with different core-shell wire ratios are woven using an 8-64 spindle loom. 8-64 silk threads with a tex of 1-10 are used as the shell threads, and 1-3 silk threads with a tex of 5-60 are used as the core threads. The shell threads and core threads are made into two-strand twisted threads with two-way twisting in opposite directions, and the twist is 400-1200Z+500-900S. According to the requirements in YY 1116-2020 standard, braided medical sutures with sizes from 12-0 to 5 are designed, and the wire diameter is 0.001-0.950mm.

[0082] (2) Degumming of silk suture: The silk suture is immersed in a sodium carbonate or sodium bicarbonate solution with a concentration of 0.01-0.06M or 0.01-0.1% (w / v), boiled in a water bath for 30-60min, rinsed with deionized water multiple times, and air-dried overnight in a fume hood.

[0083] (3) Preparation of composite serine protease solution: Proteinase K and streptococcal protease XIV or XXI are dissolved in deionized water, phosphate buffer, or Tris-HCl solution with a pH of 5-9, with a concentration of 1-300U / mL, and placed at 10-70°C for 1-30min. Subsequently, Proteinase K and streptococcal protease XIV or XXI are mixed in a ratio of 1:1-10 or 1-10:1 to obtain a composite protease treatment solution.

[0084] (4) Enzyme pretreatment of silk suture: The silk suture is soaked in the composite protease solution and pretreated in a forced-air drying oven at 10-100°C for 1-48h; after enzyme pretreatment, it is washed with deionized water until neutral.

[0085] (5) Coating with functional silk fibroin solution: 0.1-1% (by mass) of nano-zinc oxide, nano-silica, graphene, or interfacial connection substances such as chitosan, sodium alginate, and sodium hyaluronate are added to the silk fibroin solution to obtain a silk fibroin solution carrying antibacterial particles or interfacial connection substances. The enzyme-pretreated silk suture is immersed in the silk fibroin solution carrying antibacterial particles at 40-80°C for 20-60min, then dried in an oven at 100-130°C for 1-3min, and then dried at 40-60°C, or pre-frozen at -80°C for 1-6h and then freeze-dried to obtain a coated suture.

[0086] (6) Fumigation of coated suture: The dried suture is fumigated in steam at 100-120°C for 3-72h, and then vacuum degassed and dried to obtain a controllable degradation silk suture. Example 4

[0087] This example demonstrates a preparation method of a controllable degradation silk medical suture, and the specific steps are as follows:

[0088] (1) Weaving of silk suture: Two silk yarns with reverse and forward twisting are used as the outer shell yarn and the inner core yarn of the braided suture. The twist is 400 - 1200Z + 500 - 900S. The shell yarn uses 1 - 3 strands of 1 - 6tex yarn, and the inner core yarn uses 1 - 3 strands of 4 - 8tex yarn. The shell yarn and the core yarn are braided on an 8 - 64 spindle braiding machine, and the diameter of the braided suture is 0.001 - 0.950mm.

[0089] (2) Degumming of silk suture: Immerse the silk suture in a solution of papain, trypsin, or pronase at a concentration of 1 - 100U / mL. The bath ratio of the suture to the enzyme solution is 1:10 - 1000. The enzyme degumming temperature is 30 - 80°C, and the time is 10 - 60min. After degumming, wash it 1 - 3 times with deionized water at 30°C - 40°C, and then dry it.

[0090] (3) Preparation of serine protease solution: Dissolve α - chymotrypsin, proteinase K, pronase XIV or XXI in 0.01 - 1M phosphate buffer, phosphate buffer, deionized water, Tris - HCl buffer, acetic acid - sodium acetate buffer, or sodium citrate buffer with a pH of 5 - 9. Prepare it into 0.1 - 1000U / mL according to the titer and activity at 0.01 - 10mg / mL. The buffer is pre - sterilized by high - temperature and high - pressure, with a sterilization temperature of 121°C and a time of 20 - 40min. The prepared solution is placed in the dark at - 20 or - 80°C, and avoid repeated freezing and thawing during use.

[0091] (4) Enzyme pretreatment of silk suture: Immerse the degummed silk suture in phosphate buffer and ultrasonically clean it for 5 - 30min, then rinse it with deionized water and dry it. Immerse the suture in one of the serine protease solutions prepared in step (3) for pretreatment, with a bath ratio of 1:100 - 1000, a temperature of 25 - 90°C, and a time of 1 - 72h. Then immerse it in another serine protease solution, with a dosage ratio of 1:1, for pretreatment, with a bath ratio of 1:100 - 1000, a temperature of 25 - 90°C, and a time of 1 - 72h. The above treatments are placed in a forced - air oven or drying oven with air flow and apply a heat flow effect. After the pretreatment, immerse it in PBS solution and treat it at 100 - 120°C for 5 - 20min, or boil it in water bath for 30 - 60min to inactivate the protease. Wash it thoroughly with water to remove the remaining enzyme solution on the surface and air - dry it naturally.

[0092] (5) Functional silk fibroin solution coating: Add fluorescent substances rhodamine B and calcein into a silk fibroin solution with a mass percentage of 0.1 - 10%. The mass percentage of the fluorescent substance to silk fibroin is 1:10 - 1000. Mix them evenly in the silk fibroin solution to obtain a silk fibroin solution carrying the fluorescent substance. Subsequently, immerse the suture into the silk fibroin solution carrying the fluorescent substance with a bath ratio of 1:100 - 1000, soak at 25 - 90 °C for 20 - 60 min, then take it out, gently rinse with deionized water, dry at 100 - 130 °C for 1 - 3 min to improve the bonding fastness, and then dry at 40 - 80 °C for 1 - 3 h to obtain a coated suture.

[0093] (6) Fumigation and heat setting treatment of the braided suture: Put the suture into a dryer, add methanol, ethanol or deionized water into the dryer. Place the coated suture on the partition of the dryer at 25 - 120 °C and fumigate for 1 - 72 h, then vacuum remove the bubbles and dry to obtain a controllable degradation silk suture.

[0094] The test performance and analysis of the controllable degradation medical silk suture samples prepared by the above four examples can be referred to Figures 1 to 20 , Tables 1 to 6. Table 1 is the amino acid analysis result table in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (amino acid composition (mol%) of silk fibroin pretreated with 3U / mL proteinase K); Table 2 is the amino acid analysis result table in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (amino acid composition (mol%) of silk fibroin pretreated with 30U / mL proteinase K); Table 3 is the amino acid analysis result table in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (amino acid composition (mol%) of silk fibroin pretreated with 300U / mL proteinase K); Table 4 is the comparison table of the degradation mechanical properties of silk treated with two enzymes in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (mechanical properties of synergistic pretreatment of proteinase K and proteinase XIV); Table 5 is the comparison table of the degradation mechanical properties of silk treated with two enzymes in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (mechanical properties of synergistic pretreatment of proteinase K and α-chymotrypsin); Table 6 is the comparison table of the degradation mechanical properties of silk treated with two enzymes in Example 1 of the preparation method of a controllable degradation silk medical suture described in the present invention (mechanical properties of synergistic pretreatment of proteinase XXI and α-chymotrypsin).

[0095]

[0096] Table 1

[0097]

[0098] Table 2

[0099]

[0100] Table 3

[0101]

[0102] Table 4

[0103]

[0104] Table 5

[0105]

[0106] Table 6

[0107] As Figures 1 - 3 shown, with the increase of time and concentration, grooves appear on the surface of the enzyme-pretreated silk fibers, and the cracks become more obvious. In some groups, the sutures break. As Figure 4 and 5 shown, with the increase of time and concentration, the wire diameter, mass and mechanical properties of silk gradually decrease. As Figure 6 shown, with the increase of time and concentration, the content of β-sheet in the secondary structure of silk generally shows an increasing trend, but it first decreases and then increases during the increase process, indicating that the overall trend of enzymatic degradation is to degrade the amorphous region first and then the crystalline region. However, the degradation rate of the crystalline region is slower at first, then the degradation rate accelerates, and finally it slows down again. As Figure 7 shown, the X-ray diffraction results show that with the increase of time and concentration, the peak intensity at 20.5° first increases and then decreases, and the full width at half maximum first increases and then decreases, and the crystallinity first decreases and then increases. As Figure 8 shown, the decomposition temperature first decreases and then increases, reflecting that the crystallinity first decreases and then increases. As Figure 9 and Table 1-3 shown, the contents of glycine, alanine and serine (Gly, Ala, Ser) first decrease and then increase, reflecting that the content of β-sheet first decreases and then increases. At the same time, with the increase of time and concentration, the contents of hydrophilic amino acids aspartic acid and tyrosine (Asp, Tyr) gradually increase and then decrease, indicating that the degradation rate of the hydrophobic region is faster at first and then slows down. As Figure 10 shown, the position at 1667 cm -1 in the Raman spectrum remains unchanged, but the peak intensity first gradually decreases and then gradually increases, indicating that the degradation treatment will cause the content of β-sheet structure to first decrease and then increase. As Figure 11 shown, in the in vitro degradation in physiological saline for 1-4 weeks, with the prolongation of time, the grooves on the surface of silk fibers become larger and the degradation becomes more intense. As Figure 12As shown, after soaking in physiological saline for 1, 2, 3, and 4 weeks, it can be seen that during the hydrolysis process of physiological saline, the β-sheet content first increases to about 60% in the first 1-2 weeks and then decreases, indicating that the hydrolysis process is the degradation of the amorphous region first and then the crystalline region. As Figure 13 shown, after soaking in physiological saline for 1-4 weeks, the mechanical properties of the medium concentration group (30 U / mL) decreased by more than 50%, meeting the requirement of a 50% decrease in two months specified by the absorbable suture standard. As Figure 14 shown, after soaking in physiological saline for 1-4 weeks, the full width at half maximum of silk degradation gradually decreases, the crystallinity also continuously decreases, and the peak intensity gradually weakens, reflecting that with the passage of time, the crystalline region of silk gradually hydrolyzes. As Figure 15 shown, after soaking in physiological saline for 1-4 weeks, the decomposition temperature gradually decreases, while the corresponding enthalpy first increases and then decreases, reflecting that the hydrolysis process of the amorphous region leads to an increase in the content of the crystalline region, resulting in an increase in enthalpy. At the same time, the continuous decrease in the decomposition temperature indicates that the destruction of the silk hydrolysis structure leads to a decrease in the decomposition temperature. As Figure 16 shown, after soaking in physiological saline for 1-4 weeks, the position at 1667 cm in the Raman spectrum remains unchanged, but the peak intensity first gradually decreases and then gradually increases, indicating that the degradation treatment will cause the content of the β-sheet structure to first decrease and then increase, reflecting the hydrolysis of the amorphous region in the first stage and the hydrolysis of the crystalline region in the second stage during soaking in physiological saline. As -1 At this position, the peak intensity first gradually decreases and then gradually increases, indicating that the degradation treatment will cause the content of the β-sheet structure to first decrease and then increase, reflecting the hydrolysis of the amorphous region in the first stage and the hydrolysis of the crystalline region in the second stage during soaking in physiological saline. As Figure 17 shown, Figure a is the control group treated with proteinase K (30 U / mL) in Tris-HCl at pH = 8.8 for 3 hours, and Figure b is the scanning electron micrograph of the silk fibroin coating treatment. Subsequently, after soaking in physiological saline for 1-4 weeks, it can be seen that the silk fibroin coating fills the gaps in the coating. With the passage of time, the degradation gradually becomes severe, and the coating rate. As Figure 18 shown, after pretreatment with proteinase K (30 U / mL for 6 h), the silk suture drops to near the standard of the lowest requirement for absorbable sutures. Subsequently, it is compared with the knot tensile breaking strength of the silk fibroin coating, the silk fibroin coating loaded with proteinase XIV, and the methanol fumigation treatment. The silk fibroin coating delays the degradation rate in the first 1-2 weeks and is similar to the control group in the subsequent 3-4 weeks. Silk protein is a water-soluble protein that can increase mechanical properties without affecting the degradation rate. After treatment with methanol vapor for 24 hours, the mechanical properties of the silk suture further increase. The degradation rate of the silk suture after methanol fumigation is delayed compared to the untreated silk suture. After loading 1% (by mass) of proteinase XIV in the silk fibroin solution, the degradation of the silk suture after fumigation is promoted. The degradation rate can be regulated by controlling the loading amount of the protease, the fumigation time, and the amount of silk fibroin in the coating. As Figure 19 shown, after adding berberine to the silk fibroin coating, the antibacterial zone is significant, indicating that the functional coating has good antibacterial effects. As Figure 20As shown in the figure, adding calcein and rhodamine B to the silk fibroin coating to simulate the distribution of functional particles in the coating, it can be seen that the fluorescent substances are evenly distributed under the laser confocal microscope, indicating that the functional substances are evenly distributed in the coating.

[0108] In summary, the suture obtained by the preparation method of the controllable degradation silk medical suture described in the present invention has good mechanical strength, reduces the crystal crystallinity of silk, and the mechanical properties of the suture with the coating after enzyme pretreatment decrease by more than 50% after being soaked in physiological saline for 28 days, meeting the requirement that the mechanical properties of absorbable sutures decrease by 50% within two months. At the same time, the advantages of silk such as biodegradability and biocompatibility are retained, and the functional coating can endow silk with good antibacterial and degradation-promoting properties, while improving the mechanical properties. The degradation rate and mechanical properties of the suture are controllable, and the method is simple, feasible and operable.

[0109] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A preparation method of a controllable degradation silk medical suture, characterized in that, The specific steps are as follows: (1) Weave the silk shell thread and the core thread on a knitting machine to obtain a knitted suture; (2) Degum the knitted suture to obtain a degummed suture; (3) Dissolve serine protease in a buffer solution, and prepare a serine protease solution with a concentration of 0.1 - 1000 U / mL at 0.01 - 10 mg / mL. Gently shake it, avoid light and violent stirring, and age it in a hot air stream at a temperature of 20 - 80 °C for 1 - 10 min for further dissolution. Store it in the dark at -20 or -80 °C to obtain a serine protease solution, where the serine protease is protease K, or a mixture of protease K and protease XIV, or a mixture of protease K and protease XXI; (4) Immerse the degummed suture in the serine protease solution for enzyme pretreatment; (5) Immerse the suture after enzyme pretreatment in a functional silk fibroin solution with a bath ratio of 1:100 - 1000, soak it at a temperature of 25 - 90 °C for 20 - 60 min, then take it out, rinse it with deionized water, dry it at a temperature of 100 - 130 °C for 1 - 3 min, and then dry it at a temperature of 40 - 80 °C for 1 - 3 h to obtain a silk fibroin-coated suture with a relatively high content of β-sheet structure, or naturally air-dry the soaked suture in a fume hood at a temperature of 0 - 40 °C to obtain a silk fibroin-coated suture mainly composed of random coils and α-helix structures, or freeze the soaked suture at -20 °C or -80 °C for 1 - 6 h, and then lyophilize it to obtain a silk fibroin-coated suture mainly composed of easily absorbable Silk I crystal form; (6) Fumigate and thermally shape the silk fibroin-coated suture to obtain a controllable degradation silk medical suture.

2. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that: The buffer solution is any one of 0.01 - 1 M phosphate buffer solution, phosphate buffer, deionized water, Tris-HCl buffer solution, acetic acid-sodium acetate buffer solution, and sodium citrate buffer solution. The pH value range of the buffer solution is 5 - 9. The buffer solution is pre-sterilized by high-temperature and high-pressure, and the sterilization temperature is 120 - 140 °C, and the time is 20 - 40 min.

3. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that: Before step (4), first immerse the degummed suture in a phosphate buffer solution, ultrasonically clean it for 5 - 30 min, and then rinse it with deionized water and dry it.

4. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that, In step (4), the bath ratio of the degummed suture to the serine protease solution is 1:100 - 1000, and the temperature of the enzyme pretreatment is 25 - 90 °C, and the time is 1 - 72 h.

5. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that: After step (4), immerse the degummed suture in a phosphate buffer solution, treat it at a temperature of 100 - 120 °C for 5 - 20 min, or boil it in water bath for 30 - 60 min to inactivate the protease, then wash it thoroughly with water to wash away the remaining enzyme solution on the surface, and air-dry it overnight.

6. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that, In step (5), the preparation method of the functional silk fibroin solution is as follows: adding any one or more of serine protease, fluorescent substance, antibacterial drug, antibacterial particle or interfacial connection substance into a silk fibroin solution with a mass percentage of 0.1-10%, wherein the mass percentage of any one or more of the serine protease, fluorescent substance, antibacterial drug, antibacterial particle or interfacial connection substance to the silk fibroin solution is 1:10-1000, mixing evenly to obtain the functional silk fibroin solution, wherein the serine protease is any one of α-chymotrypsin, proteinase K, proteinase XXI, proteinase XIV, the fluorescent substance is rhodamine B or calcein, the antibacterial drug is any one of berberine, curcumin, penicillin, the antibacterial particle is any one of nano-zinc oxide, nano-silica, graphene, and the interfacial connection substance is any one of chitosan, sodium alginate, sodium hyaluronate.

7. The preparation method of a controllable degradation silk medical suture according to claim 1, characterized in that, In step (6), the fumigation and heat setting treatment of the silk fibroin-coated suture to obtain a controllable degradation silk medical suture specifically is: the silk fibroin-coated suture is fumigated and heat set for 1-72 h in any one of methanol vapor, ethanol vapor, and water vapor under the condition of a temperature of 25-120 °C to obtain a controllable degradation silk medical suture.

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