CN116474155A - A preparation method of controllable degradable silk medical suture - Google Patents

Abstract

The invention discloses a method for preparing a controllable degradable silk medical suture, which comprises the steps of: weaving silk shell threads and core threads on a braiding machine to obtain a braided suture; degumming treatment to obtain a degummed suture; preparing a serine protease solution; soaking the degummed suture in a serine protease solution for enzyme pretreatment; using a functional silk fibroin protein solution to coat the suture pretreated by the enzyme to obtain a coated suture; Silk medical sutures. The suture has good mechanical strength, reduces the crystallinity of silk, and meets the requirements of absorbable suture. At the same time, it retains the advantages of silk degradability and excellent biocompatibility, and the functional coating endows silk with good antibacterial and degradation-promoting properties, which makes up for the rapid loss of mechanical properties. The degradation rate and mechanical properties of the suture are controllable, and the method is simple and easy to operate.

Description

A preparation method of controllable degradable silk medical suture

technical field

The invention belongs to the technical field of biomedical materials, and relates to a preparation method of controllable degradable silk medical suture.

Background technique

Sutures are divided into absorbable sutures and non-absorbable sutures according to the absorbability of the material. Absorbable sutures avoid the trouble caused by stitch removal, and their degradation products can be excreted by human metabolism. Existing absorbable sutures usually use synthetic materials, most of which are polymerized from several monomers. The monomer fragments produced by degradation may cause swelling and local inflammation at the implantation site. Synthetic absorbable sutures tend to have a short support time and low tension. In some parts that require high tension, the strength is difficult to meet the requirements, and there is a risk of fracture. Synthetic sutures usually do not promote wound healing and repair, and the degradation products of most synthetic materials are single and may be slightly acidic, and there are certain biocompatibility problems.

Silk is composed of natural materials silk fibroin and sericin. Sericin is located in the outer layer of silk fibers and mainly plays a bonding role. Silk fibroin becomes silk fibroin after removing sericin, which 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, and a large number of hospital clinical results have proved that degummed silk sutures will not have adverse effects on the human body after implantation in human tissues, and will not cause inflammation. The main composition of silk fibroin is amino acids, among which glycine, alanine, and serine are the main amino acids that form the crystallization region, accounting for about 85% of the total amino acid molar percentage, and serine, aspartic acid, and tyrosine with hydrophilic groups account for about 30% of the total. As a natural protein, silk fibroin can promote wound healing. Compared with other synthetic sutures, it has less chance of leaving scars and has a huge market potential.

The silk suture retained more than 50% of its mechanical properties after being implanted in vivo for two months. Silk is degradable and the degradation products are amino acids. However, due to the presence of crystalline regions rich in glycine and alanine in silk fibroin, the ability to resist protease degradation is strong, so the degradation time is longer. In order to speed up the degradation of silk sutures, researchers mainly regulated through methods such as wet spinning, degumming, coating, and genetic engineering, and explored and obtained silk sutures with accelerated degradation. For example: use sodium carbonate, sodium bicarbonate, calcium chloride to degumming the woven silk suture, and then coat a kind of absorbable suture of silk fibroin solution (CN109385710A), but the suture prepared by this method only uses the method of degumming to remove the sericin on the surface of silk, and then coats it with silk fibroin solution, which cannot damage the crystallization area of silk fibroin. Solution to obtain braided support (CN109758262A), this method does not cause damage to silk fibroin crystallization area, and coating collagen cannot promote degradation. Another example is the degradable absorbable suture (CN115192764A) obtained by preparing the spinning solution with sodium alginate and casein through wet spinning, etc., but the artificial regenerated silk obtained by wet spinning has an unstable structure, and its mechanical properties are not as good as natural silk, and the suture has a single composition, and its components are not easy to control; another example is through genetic engineering. The silkworm heavy chain recombinant protein gene and proteinase K, α-I collagenase enzyme cleavage sequences are used to identify the design gene, and then the biological fermentation method is expressed to obtain silk fibroin, and then obtained by wet method Spinning to prepare regenerated sutures (CN112301495A and CN112301496A). On the one hand, the regenerated silk obtained by wet spinning has poor mechanical strength, toughness and ductility than silk. On the other hand, gene design is difficult and costly, and the practical feasibility is not high. In the above methods, there are often problems such as undamaged silk crystallization regions, unsatisfactory structure and mechanical properties of regenerated silk fibroin, and incomparable biodegradability and biocompatibility of materials, etc., which require further improvement of preparation methods and processes. In order to overcome the defects of the prior art and solve the above problems, it is necessary to develop a new method for preparing controllable degradable silk medical sutures.

Therefore, in order to solve the problems of slow degradation of existing silk sutures, low mechanical strength of sutures obtained by methods such as regenerated silk, single components, and difficulty in controlling the degradation rate, it is necessary to prepare a controllable degradable silk medical suture.

Contents of the invention

The object of the present invention is to provide a method for preparing controllable degradable silk medical sutures to solve the above problems.

Technical scheme of the present invention is:

A preparation method of controllable degradable silk medical suture, specifically comprising:

(1) braiding the silk shell thread and the core thread on a braiding machine to obtain a braided suture thread;

(2) degumming the braided suture to obtain a degummed suture;

(3) preparing serine protease solution;

(4) soaking the degummed suture in the serine protease solution for enzyme pretreatment;

(5) Using a functional silk fibroin solution to coat the suture pretreated by the enzyme to obtain a coated suture;

(6) Fumigate and heat-set the coated suture to obtain controllable degradable silk medical suture.

Further, in step (3), the preparation of the serine protease solution specifically includes: dissolving the serine protease in the buffer solution, and preparing a 0.1-1000 U/mL serine protease solution according to the potency and activity of 0.01-10 mg/mL, shaking gently, avoiding light and vigorous stirring, aging in a hot air stream at a temperature of 20-80°C for 1-10 minutes to further dissolve, and storing in the dark at -20 or -80°C to obtain the serine protease solution.

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

Further, the buffer is any one of 0.01-1M phosphate buffer, phosphate buffer, deionized water, Tris-HCl buffer, acetic acid-sodium acetate buffer, and sodium citrate buffer. The pH value of the buffer is in the range of 5-9. The buffer is sterilized by high temperature and high pressure in advance, and the sterilization temperature is 120-140° C. for 20-40 minutes.

Further, before step (4), the degummed suture is first soaked in phosphate buffer solution, ultrasonically cleaned for 5-30 min, and then rinsed with deionized water and dried.

Further, in step (4), the serine protease solution is selected from any two serine protease solutions in α-chymotrypsin solution, protease K solution, protease XXI solution, and protease XIV solution, and mixed and co-processed in a ratio of 1:1-10 or 1-10:1, or the serine protease solution is selected from proteinase K solution, or after soaking in a serine protease solution in α-chymotrypsin, protease K, protease XXI, and protease XIV, Then soak in another serine protease solution among α-chymotrypsin, protease K, protease XXI and protease XIV for 1-72h, the bath ratio is 1:100-1000, 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 1-72h.

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

Further, 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 interface connecting substance to the silk fibroin protein 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 interface connecting substance and the silk fibroin solution is 1:10-1000, and mixing uniformly to obtain a functional silk fibroin protein solution, wherein, The serine protease is any one of α-chymotrypsin, protease K, protease XXI, and protease XIV; the fluorescent substance is rhodamine B or calcein; the antibacterial drug is any one of berberine, curcumin, and penicillin; the antibacterial particles are any one of nano-zinc oxide, nano-silica, and graphene;

Further, in step (5), the method of using the functional silk fibroin solution to coat the suture pretreated by the enzyme to obtain the coated suture specifically includes: immersing the suture pretreated by the enzyme into the functional silk fibroin solution at a bath ratio of 1:100-1000, soaking it at a temperature of 25-90°C for 20-60min, then removing it, washing it with deionized water, and drying it at a temperature of 100-130°C for 1-3 minutes. min, and then dried at 40-80°C for 1-3 hours to obtain silk fibroin-coated sutures with high structural β-fold content, or air-dried the soaked sutures at a temperature of 0-40°C to obtain silk fibroin-coated sutures with mainly random curls and α-helical structures, or put the soaked sutures at -20°C or -80°C for 1-6 hours, and then freeze-dried to obtain easily absorbable Silk I crystal form-based silk Protein-coated sutures.

Further, in step (6), the fumigation and heat setting treatment of the coated suture to obtain the controllable degradable silk medical suture specifically includes: the coated suture is fumigated and heat-set in any one of methanol steam, ethanol steam, and water vapor at a temperature of 25-120°C for 1-72 hours to obtain the controllable degradable medical silk suture.

The invention provides a method for preparing controllable degradable silk medical suture, which has the following advantages:

(1) The present invention uses a serine protease solution, which can act on major amino acids such as glycine, alanine, and serine on the crystallization region sequence of silk in a targeted manner, regulate the size and orientation structure of the crystallization region, and promote degradation. The enzymatic reaction has strong specificity, high efficiency, and mild action conditions. The degree of degradation can be adjusted by controlling the concentration, temperature, pH, and time. The process is simple and the operation is simple, and the protease can be removed by soaking, washing and other treatments.

(2) The present invention adopts silk composite weaving molding technology, by selecting positive and negative twisted yarns as the weaving material, and using shell threads and core threads of different linear densities for composite braiding, using thinner yarn shell threads as the braiding material, and relatively thicker yarns as the core thread support, shell-core composite braiding molding, and further improving mechanical properties; the wire diameter and knot breaking strength of the prepared controllable degradable silk medical suture meet the requirements of the absorbable suture standard YY 1116-2020.

(3) The present invention adopts water-soluble silk fibroin coating, fumigation, freeze-drying, and heat-setting treatment to obtain a silk fibroin coating with α-helix, random coil or β-sheet structure, and the coating can be filled into the cracks and defects treated by silk enzyme to make up for the lack of 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 of water-soluble proteins, which further compensates for the loss of mechanical properties of enzymatic degradation treatment. Silk fibroin is naturally green and environmentally friendly, non-toxic, harmless, safe and superior in performance. Adding functional substances to further accelerate degradation, endow antibacterial function and improve mechanical properties has a good application prospect.

(4) The method of the present invention is efficient and practical, and the controllable degradable 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 guides, etc. woven by controllable degradable silk, which is a new controllable degradation method.

Description of drawings

Fig. 1 is a scanning electron microscope topography (x50) of a silk suture that is degraded by protease K in the preparation method of a controllable degradable silk medical suture according to the present invention in Example 1;

Fig. 2 is a scanning electron microscope topography (x1000) of a silk suture that is degraded by proteinase K in the preparation method of a controllable degradable silk medical suture according to the present invention in Example 1;

Fig. 3 is a scanning electron microscope topography (x5000) of a silk suture that is degraded by proteinase K in the preparation method of a controllable degradable silk medical suture according to the present invention in Example 1;

Fig. 4 is a graph showing the change in wire diameter and quality in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 5 is a diagram of the variation of knotting tensile breaking strength in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 6 is the infrared spectrogram and fitting result diagram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 7 is an X-ray diffraction diagram and a fitting result diagram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 8 is a differential scanning calorimetry diagram and a fitting result diagram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 9 is a graph showing the results of amino acid analysis in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 10 is a Raman spectrogram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 11 is a scanning electron micrograph of in vitro simulated degradation in Example 1 of a preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 12 is an in vitro simulated degradation infrared spectrogram and a fitting result diagram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 13 is a graph showing the change in tensile strength of a controllable degradable silk medical suture in Example 1 in vitro simulated degradation and knotting;

Fig. 14 is an in vitro simulated degradation X-ray diffraction diagram and a fitting result diagram in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 15 is a differential scanning calorimetry diagram and a fitting result diagram of in vitro simulated degradation in Example 1 of a preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 16 is a Raman spectrogram of the in vitro simulated degradation in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 17 is a scanning electron micrograph of coating and in vitro simulated degradation in Example 1 of a preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 18 is a graph showing the change in tensile breaking strength of coating and in vitro simulated degradation knotting in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 19 is a drug-loaded antibacterial effect diagram of the coating in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention;

Fig. 20 is a diagram showing the distribution of fluorescent substances coated in Example 4 of a controllable degradable silk medical suture preparation method according to the present invention.

Detailed ways

The invention draws on the idea of twisting the "steel cables" of the suspension bridge to improve the mechanical properties of silk sutures, and the yarns with positive and negative twisted structures will stretch and shrink with the change of stress, so as to meet the requirements of passing through skin and tissues. The original silk is etched by enzyme pretreatment, the crystal structure and some properties of the original silk are retained, and the crystallization area is partially destroyed to promote the hydrolysis of the silk suture. The biocompatibility and biodegradability of the material are better than synthetic materials. On the basis of enzyme pretreated silk suture as "steel cable", the soluble silk fibroin solution is used as a functional coating to fill the pores and cracks formed by enzyme pretreatment, and the mechanical properties are improved through coating, fumigation, freeze-drying, and heat setting. Antibacterial particles, antibacterial drugs, fluorescent substances, and serine protease are added to the coating to modify silk sutures and regulate mechanical properties and degradation rates to achieve "broken and continuous" effects. The medical silk suture obtained by this method promotes degradation and maintains good mechanical properties, and can be expanded to application scenarios such as braided degradable artificial tendon/ligament scaffolds, heart valves, and nerve guides.

In some embodiments of the present invention, silk yarns are used as raw materials, and a plurality of silk shell threads and core threads of different linear densities are used to weave suture threads. Degrade silk fibroin with serine protease that hydrolyzes amino acids with strong hydrophobicity faster, reduce the structural content of crystallized regions in silk, and accelerate degradation. The silk fibroin solution coating with a concentration of 0.1-10% by mass is selected. If the concentration is too low, the mechanical improvement will not be significant, and if the concentration is too high, it will easily form a gel; and protease is added to the coating solution to improve the degradability of the coating, antibacterial particles and antibacterial drugs are added to give antibacterial function, and interface connecting substances and silk fibroin coating are added to form hydrogen bond cross-linking, which further improves the coating fastness and enhances mechanical properties.

The specific operation steps of the preparation method of controllable degradable silk medical suture are as follows:

S1, braiding the silk shell thread and the core thread on a braiding machine to obtain a braided silk suture thread.

Two 1-3 strands of 1-10tex silk shell yarn and 1-3 strands of 4-60tex silk core yarn with twist of 400-1200Z+500-900S are used to weave on an 8-64 spindle type braiding machine to obtain a braided silk suture.

S2, degumming the braided silk suture, followed by washing and drying to obtain a degummed suture.

Degumming the suture in 0.01-0.06M or 0.01-0.1% (w/v) sodium carbonate, sodium bicarbonate solution at 50-100°C or 1-100U/mL papain, trypsin, pronase solution at 30-80°C for 10-60min, the bath ratio of suture and degumming solution is 1:10-1000, rinse with deionized water 1-3 times, and then dry to obtain degumming sutures.

S3, preparing a serine protease solution.

Dissolve α-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (Candida albicans), protease XXI (streptococcus griseus), and protease XIV (streptococcus griseus) in 0.01-1M phosphate buffer, pH=5-9 phosphate buffer, phosphate buffer, deionized water, Tris-HCl buffer, acetic acid-sodium acetate buffer, which have been sterilized at 120-140°C for 20-40min Or in sodium citrate buffer solution, according to the potency and activity, prepare 0.1-1000U/mL serine protease solution according to 0.01-10mg/mL, shake gently, avoid light and vigorous stirring, age in a hot air flow at 20-80°C for 1-10min to further dissolve, and store at -20 or -80°C in the dark to obtain a serine protease solution. Avoid repeated freezing and thawing during use.

S4, soaking the degummed suture in the serine protease solution to perform enzyme pretreatment.

Soak the degummed suture in phosphate buffer, ultrasonically clean it for 5-30min, rinse it with deionized water, dry it, soak it in 0.1-1000U/mLα-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (Candida albicans), protease XXI (streptococcus griseus), protease XIV (streptococcus griseus) at a ratio of 1:1-10 or 1-10:1 at 25-90°C Bacteria) in two different serine protease solutions above, or in α-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (Candococcus limberii), protease XXI (streptococcus griseus), protease XIV (streptococcus griseus) in a serine protease solution for 1-72h and then in α-chymotrypsin (bovine pancreas, porcine pancreas), proteinase K (candida albicans limberii), protease XXI (gray streptococcus), protease XIV (streptococcus griseus) in another solution soaked for 1-72h, bath ratio is 1:100-1000, because heat flow is required during enzyme pretreatment, placed in a blast oven or drying oven with heat flow. Then soak the silk suture in phosphate buffer, treat it at 100-120°C for 5-20 minutes or boil it in a water bath for 30-60 minutes to inactivate the protease, then fully wash with water to remove the remaining enzyme solution on the surface, and air-dry overnight.

S5, using the functional silk fibroin solution to impregnate, coat and dry the enzyme-pretreated suture to obtain a coated suture.

Add serine protease α-chymotrypsin (bovine pancreas, porcine pancreas), protease K (candida albicans), protease XXI (streptococcus griseus), protease XIV (streptococcus griseus), or fluorescent substance rhodamine B, one of calcein, or antibacterial drugs such as berberine, curcumin, one of penicillin, or antibacterial particles nano-zinc oxide, nano-di One of silicon oxide and graphene, or one of interface linking substances chitosan, sodium alginate, sodium hyaluronate, the above substances: silk fibroin solution (mass percentage) = 1:10-1000, stirring evenly to obtain a functional silk fibroin solution. Soak the suture thread after enzyme pretreatment in the functional silk fibroin solution with a bath ratio of 1:100-1000, soak for 20-60min at 25-90°C, remove and gently rinse with deionized water. Dry at 100-130°C for 1-3min to improve the bonding fastness, dry at 40-80°C for 1-12h, and dry to obtain silk fibroin-coated sutures with high β-fold structure content, or take out the coated sutures after soaking and place them in a fume hood at 0-40°C to dry naturally to obtain silk fibroin-coated sutures with irregular curls and α-helical structures, or take out the coated sutures and place them in -20°C or -80°C freezer for 1-6 h, followed by lyophilization to obtain silk fibroin-coated sutures mainly in the easily absorbable Silk I crystal form.

S6, fumigate and heat-set the coated suture to obtain a controllable degradable silk suture.

The coated suture is fumigated under methanol vapor, ethanol vapor or water vapor at 25-120°C, and heat-set for 1-72 hours to promote the random curl and α-helix formation of β-fold of water-soluble silk fibroin in the coating to compensate for mechanical loss, and obtain controllable degradation of silk medical suture.

In order to make the above objects, features and advantages of the present invention more obvious and comprehensible, the technical solution of the present invention will be further described below in conjunction with specific examples. But the present invention is not limited to the listed embodiments, but also includes any other known changes within the claimed scope of the present invention.

Reference herein to "one embodiment" or "an embodiment" refers to a particular feature, structure or characteristic that can be included in at least one implementation of the present invention. "In one embodiment" appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.

Example 1

This example shows a preparation method of controllable degradable silk medical suture, the specific steps are as follows:

(1) Weaving of silk suture: use 8-64 spindle weaving machine to weave fibers with different proportions of shell and core wires. Two strands of twisted silk with a twist of 400-1200Z+500-900S are used for bi-directional twisted silk weaving, 1-10tex silk is used as the shell thread, and 5-60tex silk is used as the core thread. According to the requirements of the YY 1116-2020 standard, design 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, 5 braided medical suture thread, with a diameter of 0.001-0.950mm.

(2) Degumming of silk sutures: immerse silk sutures in 0.01-1g/L sodium carbonate solution, treat in a slightly boiling state for 30-60min, rinse with deionized water several times, and air-dry overnight in a fume hood.

(3) Preparation of serine protease solution: mix serine protease K and protease XIV at 1:1-10 or 1-10:1, dissolve in deionized water, phosphate buffer or Tris-HCl solution with pH=5-9 at a concentration of 1-300 U/mL, and place at 10-70°C for 1-30min.

(4) Enzyme pretreatment of silk sutures: Soak the degummed sutures in a mixed solution of proteinase K and proteinase XIV at a bath ratio of 1:10-1000, and treat them in a blast drying oven at 10-100°C for 1-48 hours.

(5) Functional silk fibroin solution coating: add 0.1-1% by mass of berberine, curcumin, and penicillin to the silk fibroin solution to obtain a drug-loaded silk fibroin solution or add protease XIV, protease XXI, protein K, and α-chymotrypsin to obtain a silk fibroin solution carrying serine protease. Soak the enzyme-pretreated silk suture in the drug-loaded or protease-loaded silk fibroin solution at 40-80°C for 20-60 minutes, then dry at 100-130°C for 1-3 minutes, then dry at 40-60°C, pre-freeze at -80°C for 1-6 hours, and then freeze-dry to obtain a coated suture.

(6) Fumigation of the coated suture: place the dried suture in methanol vapor, ethanol vapor or water vapor and fumigate at 40-100° C. for 3-24 hours to obtain a controllably degradable silk suture.

Example 2

This example shows a preparation method of controllable degradable silk medical suture, the specific steps are as follows:

(1) Silk yarn degumming: the shell thread of the suture thread adopts two strands of 1-10tex silk yarn, and the core thread adopts two strands of 5-60tex silk yarn. Add 10-20L of distilled water to the water bath, boil, add 0.1-10g of sodium carbonate, the concentration is 0.01-1mol/L, immerse the silk in the solution, boil for 30-60min, rinse in distilled water, and dry overnight at room temperature.

(2) Weaving of silk sutures: weave the degummed silk yarns on a knitting machine with 8-64 spindles to obtain sutures with a wire diameter of 0.001-0.950mm and mechanical properties meeting the requirements of the standard YY 1116-2020.

(3) Preparation of serine protease solution: mix pronase XIV or XXI and protease 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 1-100mg/L, and the concentration is 1-300U/mL, and aged at a temperature of 30-50°C for 1-30min.

(4) Silk suture enzyme pretreatment: immerse the degummed silk suture in a mixed solution of pronase protease and proteinase K at a bath ratio of 1:10-1000, put it in an oven at 10-100°C, blow nitrogen or carbon dioxide into the surrounding environment of the sample at a flow rate of 200-1000mL/min, treat for 1-48h, and shake gently every 30-60min to make the silk suture fully and evenly contact with the protease solution.

(5) Coating of functional silk fibroin solution: adding 0.1-1% pronase protease to the silk fibroin solution to obtain a degradable silk fibroin solution. Soak the pronase-pretreated silk suture at 20-30°C for 20-60 minutes in the degradative silk fibroin solution, then gently rinse with distilled water, and repeat the soaking and rinsing for 1-20 times. Then freeze-dry after pre-freezing at -20°C or -80°C for 1-6h to obtain coated sutures.

(6) Fumigation of the coated suture: fumigate the dried suture in water vapor at 100-120° C. for 3-72 hours, and dry it in vacuum to obtain a controllable degradable silk suture.

Example 3

This example shows a preparation method of controllable degradable silk medical suture, the specific steps are as follows:

(1) Weaving of silk suture: 8-64 spindle braiding machines are used to weave fibers with different ratios of shell and core wires, 8-64 silks of 1-10tex are used as shell threads, 1-3 silks of 5-60tex are used as core threads, and shell threads and core threads are made of 2 strands twisted in both forward and reverse directions, with a twist of 400-1200Z+500-900S. According to the requirements in the YY 1116-2020 standard, design 12-0 to 5 braided medical sutures with a diameter of 0.001-0.950mm.

(2) Degumming of silk suture: immerse silk suture in 0.01-0.06M or 0.01-0.1% (w/v) sodium carbonate and sodium bicarbonate solution, boil in a water bath for 30-60min, rinse with deionized water several times, and air-dry overnight in a fume hood.

(3) Preparation of compound serine protease solution: Dissolve proteinase K and pronase XIV or XXI in deionized water, phosphate buffer or Tris-HCl solution with pH=5-9 at a concentration of 1-300 U/mL, and place at 10-70°C for 1-30min. Then proteinase K and pronase XIV or XXI are mixed at 1:1-10 or 1-10:1 to obtain a complex protease treatment solution.

(4) Enzyme pretreatment of silk sutures: Soak silk sutures in a compounded protease solution and pretreat them in a blast drying oven at 10-100° C. for 1-48 hours; after enzyme pretreatments, wash them with deionized water until neutral.

(5) Functional silk fibroin solution coating: add 0.1-1% by mass of nano-zinc oxide, nano-silicon dioxide, graphene, or interface linking substances chitosan, sodium alginate, sodium hyaluronate to the silk fibroin solution to obtain a silk fibroin solution carrying antibacterial particles or interface linking substances. Soak the enzyme-pretreated silk suture at 40-80°C for 20-60 minutes in the silk fibroin protein solution loaded with antibacterial particles, then bake it at 100-130°C for 1-3 minutes, then place it at 40-60°C for drying, or pre-freeze at -80°C for 1-6 hours and then freeze-dry to obtain a coated suture.

(6) Fumigation of the coated suture: Fumigate the dried suture in water vapor at 100-120° C. for 3-72 hours, and vacuum degassing and drying to obtain a controllable degradable silk suture.

Example 4

This example shows a preparation method of controllable degradable silk medical suture, the specific steps are as follows:

(1) Weaving of silk sutures: 2 silk yarns twisted in forward and reverse directions are used as the outer shell thread and inner core thread of the braided suture, with a twist of 400-1200Z+500-900S. The shell thread adopts 1-3 strands of 1-6tex yarn, and the inner core wire adopts 1-3 strands of 4-8tex yarn. The shell wire and the core wire are braided on an 8-64 spindle braiding machine, and the diameter of the braided suture thread is 0.001-0.950mm.

(2) Degumming of silk suture: immerse silk suture in 1-100U/mL papain, trypsin, pronase solution, the bath ratio of suture and enzyme solution is 1:10-1000, the enzyme degumming temperature is 30-80°C, and the time is 10-60min. After degumming, use 30°C-40°C deionized water to wash 1-3 times, and then dry.

(3) Preparation of serine protease solution: Dissolve α-chymotrypsin, protease K, pronase XIV or XXI in 0.01-1M phosphate buffer, phosphate buffer, deionized water, Tris-HCl buffer, acetic acid-sodium acetate buffer, sodium citrate buffer, pH=5-9, according to titer and activity according to 0.01-10mg/mL to configure 0.1-1000U/mL. The buffer solution is pre-sterilized by high temperature and high pressure at 121° C. for 20-40 minutes. Store the prepared solution away from light at -20 or -80°C, and avoid repeated freezing and thawing during use.

(4) Enzyme pretreatment of silk sutures: Soak the degummed silk sutures in phosphate buffer solution, ultrasonically clean them for 5-30 minutes, then rinse them with deionized water and dry them. Soak the suture in one of the serine protease solutions prepared in the above step (3) for pretreatment, the bath ratio is 1:100-1000, the temperature is 25-90°C, and the time is 1-72h, and then immersed in another serine protease solution, the dosage ratio of the two is 1:1, and the pretreatment is carried out, the bath ratio is 1:100-1000, the temperature is 25-90°C, and the time is 1-72h. The above treatments are placed in a drum with airflow In the air oven or drying box, heat flow is applied. After pretreatment, soak in PBS solution, treat at 100-120°C for 5-20 minutes, or boil in a water bath for 30-60 minutes to inactivate protease. Fully wash with water to remove residual enzyme solution on the surface, and air dry naturally.

(5) Coating with functional silk fibroin solution: add fluorescent substances rhodamine B and calcein to 0.1-10% silk fibroin solution by mass percentage, the mass percentage is fluorescent substance: silk fibroin=1:10-1000, and mix them evenly in the silk fibroin solution to obtain a silk fibroin solution carrying fluorescent substances. Then immerse the suture in the silk fibroin solution loaded with fluorescent substances, the bath ratio is 1:100-1000, soak at 25-90°C for 20-60min, then remove it, rinse gently with deionized water, dry at 100-130°C for 1-3min to improve the binding fastness, dry at 40-80°C for 1-3h, and dry to obtain a coated suture.

(6) Fumigate and heat-set the braided suture: put the suture in a desiccator, add methanol, ethanol or deionized water into the desiccator, put the coated suture on the separator of the desiccator at 25-120°C, fumigate for 1-72 hours, remove air bubbles and dry under vacuum to obtain controllable degradable silk suture.

For the test performance and analysis of the controllable degradable medical silk suture samples made from the above four examples, please refer to Fig. 1 to Fig. 20 and Table 1 to Table 6. Table 1 is the amino acid analysis result table in Example 1 of the preparation method of a controllable degradable silk medical suture according to the present invention (the amino acid composition (mol%) of silk fibroin protein pretreated with 3 U/mL proteinase K); Table 2 is the amino acid analysis result table of the preparation method of a controllable degradable silk medical suture according to the present invention in Example 1 (amino acid composition (mol%) of silk fibroin protein pretreated with 30 U/mL proteinase K); Table 3 is a controllable degradable silk medical suture according to the present invention The preparation method of suture is in the amino acid analysis results table in Example 1 (300U/mL protease K pretreated silk fibroin protein amino acid composition (mol%)); Table 4 is a comparison table of the degradation mechanical properties of a controllable degradable silk medical suture in Example 1 using two enzymes to co-treat silk (protease K and protease XIV synergistic pretreatment mechanical properties); Table 6 is a comparison table of degradation mechanical properties of processed silk (protease K and α-chymotrypsin synergistic pretreatment mechanical properties); Table 6 is a comparison table of degradation mechanical properties of silk treated with two enzymes in Example 1 for a preparation method of a controllable degradable silk medical suture according to the present invention (protease XXI and α-chymotrypsin synergistic pretreatment mechanical properties).

Table 1

Table 2

table 3

Table 4

table 5

Table 6

As shown in Figures 1-3, with the increase of time and concentration, grooves appeared on the surface of silk fibers pretreated with enzymes, the cracks became more obvious, and some groups of sutures broke. As shown in Figures 4 and 5, with the increase of time and concentration, the wire diameter and quality of silk gradually decrease, and the mechanical properties gradually decrease. As shown in Figure 6, with the increase of time and concentration, the β-sheet content of the secondary structure of silk showed an overall increasing trend, but decreased first and then increased during the increase process, indicating that the overall trend of enzymatic degradation was to degrade the amorphous region first, and then degrade the crystalline region. However, the degradation rate of the crystalline region was slow first, then the degradation speed was accelerated, and finally there was a trend of slowing down. As shown in Figure 7, 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, the combined half-width height first increases and then decreases, and the crystallinity first decreases and then increases. As shown in Figure 8, the decomposition temperature first decreases and then increases, reflecting that the crystallinity first decreases and then increases. As shown in Figure 9 and Tables 1-3, the contents of glycine, alanine, and serine (Gly, Ala, Ser) first decreased and then increased, reflecting that the β-sheet content first decreased and then increased. At the same time, with the increase of time and concentration, the content of amino acids aspartic acid and tyrosine (Asp, Tyr) in the hydrophilic region gradually increased, and then decreased. The rate of degradation of the hydrophobic region was faster first, and then slowed down. As shown in Figure 10, the position at 1667cm ^-1 in the Raman spectrum remains the same, but the peak intensity first gradually decreases and then gradually increases, which indicates that the degradation treatment will lead to the first decrease and then increase of the β-sheet structure content. As shown in Figure 11, after 1-4 weeks of in vitro degradation in saline simulation, as time goes on, the grooves on the surface of silk fibers become larger and the degradation becomes more severe. As shown in Figure 12, after soaking in normal saline for 1, 2, 3, and 4 weeks, it can be seen that during the hydrolysis process of normal saline, the β-sheet content first increased to about 60% in 1 and 2 weeks, and then decreased, indicating that the hydrolysis process is the degradation of the amorphous region first, followed by the degradation of the crystalline region. As shown in Figure 13, after soaking in normal saline for 1-4 weeks, the mechanical properties of the medium-concentration group (30U/mL) decreased by more than 50%, which met the requirement of 50% decrease in two months stipulated in the absorbable suture standard. As shown in Figure 14, after soaking in normal saline for 1-4 weeks, the degraded half-width height of silk gradually decreased, the degree of crystallinity also decreased, and the peak intensity gradually weakened, reflecting that the crystallization area of silk was gradually hydrolyzed as time went on. As shown in Figure 15, after soaking in normal saline for 1-4 weeks, the decomposition temperature gradually decreased, while the corresponding enthalpy first increased and then decreased, reflecting the degradation process. The hydrolysis of the amorphous region led to an increase in the content of the crystallization region, and the increase in enthalpy. At the same time, the continuous decrease in the decomposition temperature indicated that the destruction of the hydrolyzed structure of silk resulted in a decrease in the decomposition temperature. As shown in Figure 16, after soaking in normal saline for 1-4 weeks, the position at 1667 cm ^-1 in the Raman spectrum remains unchanged, but the peak intensity first gradually decreases and then gradually increases, which indicates that the content of β-sheet structure decreases first and then increases after degradation treatment, reflecting the hydrolysis of the amorphous region in the first stage and the hydrolysis of the crystalline region in the second stage after immersion in physiological saline. As shown in Figure 17, Figure a is the control group treated with proteinase K (30U/mL) in Tris-HCl at pH = 8.8 for 3 hours, and Figure b is the scanning electron microscope image of silk fibroin coating treatment, followed by soaking in normal saline for 1-4 weeks, it can be seen that the silk fibroin coating fills the gaps in the coating. As shown in Figure 18, after proteinase K pretreatment (30U/mL 6h), silk sutures dropped to the minimum standard for absorbable sutures, and then compared with silk fibroin coatings, silk fibroin coatings equipped with protease XIV, and methanol fumigation treatment for knot tensile breaking strength, silk fibroin coatings delayed the degradation rate in 1-2 weeks, and then 3-4 weeks were similar to the control group. Silk protein is a water-soluble protein that can increase mechanical properties without affecting the degradation rate; The mechanical properties of the silk suture were further improved, and the degradation rate of the suture after methanol fumigation was delayed compared with that of the untreated silk suture. After carrying protease XIV in the silk fibroin solution with a mass percentage of 1% of the silk fibroin solution, the degradation of the silk suture after fumigation was promoted. The degradation rate can be regulated by controlling the amount of protease carried, the fumigation time and the amount of silk fibroin in the coating. As shown in Figure 19, after adding berberine to the silk fibroin coating, the antibacterial zone is significant, indicating that the functional coating has a good antibacterial effect. As shown in Figure 20, calcein and rhodamine B were added 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.

In summary, the method for preparing controllable degradable silk medical suture according to the present invention has good mechanical strength of the suture, which reduces the crystallinity of silk, and the mechanical properties of the coated suture after enzymatic pretreatment are reduced by more than 50% after soaking in normal saline for 28 days, which meets the requirement that the mechanical properties of absorbable sutures decrease by 50% within two months. While retaining the advantages of silk degradability and biocompatibility, the functional coating can endow silk with good antibacterial and degradation-promoting properties, while improving mechanical properties. The degradation rate and mechanical properties of the suture are controllable, and the method is simple, easy and operable.

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. 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, which should be covered by the scope of the claims of the present invention.

Claims (10)

1. A preparation method of a controllable degradable silk medical suture is characterized by comprising the following specific steps:

(1) Weaving the silk shell thread and the core thread on a weaving machine to obtain a woven suture;

(2) Degumming the braided suture line to obtain an degummed suture line;

(3) Preparing serine protease solution;

(4) Immersing the degummed suture in the serine protease solution for enzyme pretreatment;

(5) Coating the suture after enzyme pretreatment by adopting a functional silk fibroin solution to obtain a coated suture;

(6) And fumigating and heat setting the coated suture line to obtain the controllable degradable silk medical suture line.

2. The method for preparing a medical suture of controllably degradable silk according to claim 1, wherein in step (3), the preparation of serine protease solution is specifically: dissolving serine protease in buffer solution, preparing 0.1-1000U/mL serine protease solution according to 0.01-10mg/mL serine protease solution, gently shaking, avoiding illumination and intense stirring, aging in hot air flow at 20-80deg.C for 1-10min for further dissolution, and storing at-20 or-80deg.C in dark place to obtain serine protease solution.

3. The method for preparing the controllable degradable silk medical suture line according to claim 2, which is characterized in that: the serine protease is any one of alpha-chymotrypsin, proteinase K, proteinase XXI and proteinase XIV.

4. The method for preparing the controllable degradable silk medical suture line according to claim 2, which is characterized in that: the buffer solution is any one of 0.01-1M phosphate buffer solution, phosphoric acid 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 sterilized at high temperature and high pressure in advance, the sterilizing temperature is 120-140 ℃ and the time is 20-40min.

5. The method for preparing the controllable degradable silk medical suture line according to claim 2, which is characterized in that: before the step (4), the degummed suture is soaked in phosphate buffer solution, ultrasonically cleaned for 5-30min, and then washed by deionized water and dried.

6. A method of preparing a controllably degradable silk medical suture according to claim 3, wherein in step (4), the serine protease solution is selected from any two serine protease solutions of alpha-chymotrypsin solution, proteinase K solution, proteinase XXI solution and proteinase XIV solution, and the serine protease solution is mixed and treated cooperatively according to the ratio of 1:1-10 or 1-10:1, or the serine protease solution is selected from proteinase K solution, or is soaked in one serine protease solution of alpha-chymotrypsin, proteinase K, proteinase XXI and proteinase XIV before being soaked in the other serine protease solution of alpha-chymotrypsin, proteinase K, proteinase XXI and proteinase XIV, 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 ℃ for 1-72h.

7. The method for preparing the controllable degradable silk medical suture line according to claim 1, which is characterized in that: after the step (4), soaking the degummed suture in phosphate buffer solution, treating at 100-120 ℃ for 5-20min, or boiling in water bath for 30-60min, inactivating protease, then fully washing, washing off the enzyme solution remaining on the surface, and air-drying overnight.

8. The method for preparing a medical suture of controllably degradable silk according to claim 1, wherein in step (5), the method for preparing the functional silk fibroin solution comprises: adding any one or more of serine protease, fluorescent substance, antibacterial particles or interface connection substances into 0.1-10% of silk fibroin solution, uniformly mixing any one or more of serine protease, fluorescent substance, antibacterial drug, antibacterial particles or interface connection substances with the silk fibroin solution in a mass ratio of 1:10-1000, and obtaining a functional silk fibroin solution, wherein the serine protease is any one of alpha-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 particles are any one of nano zinc oxide, nano silicon dioxide and graphene, and the interface connection substances are any one of chitosan, sodium alginate and sodium hyaluronate.

9. The method for preparing a controllable degradable silk medical suture according to claim 1, wherein in the step (5), the suture coating after the enzyme pretreatment is treated by adopting the functional silk fibroin solution, and the obtained coated suture is specifically: immersing the suture subjected to enzyme pretreatment into the functional silk fibroin solution, wherein the bath ratio is 1:100-1000, immersing for 20-60min at the temperature of 25-90 ℃, then fishing out, washing with deionized water, drying for 1-3min at the temperature of 100-130 ℃, and then drying for 1-3h at the temperature of 40-80 ℃ to obtain a silk fibroin coating suture with higher beta-sheet content of the structure, or naturally air-drying the immersed suture at the temperature of 0-40 ℃ in a fume hood to obtain a silk fibroin coating suture with random curling and alpha-helical structures as main, or freezing the immersed suture at the temperature of-20 ℃ or-80 ℃ for 1-6h, and then freeze-drying to obtain a silk fibroin coating suture with easily-absorbed silk I crystal forms as main.

10. The method for preparing a controllable degradable silk medical suture according to claim 1, wherein in the step (6), the coating suture is fumigated and heat-set to obtain the controllable degradable silk medical suture specifically comprising: the coated suture line is fumigated in any one of methanol steam, ethanol steam and water steam for 1-72 hours under the condition that the temperature is 25-120 ℃ to obtain the controllable degradable silk medical suture line.