WO2021068743A1 - 一种自支撑丝素蛋白导管支架的成型方法 - Google Patents

一种自支撑丝素蛋白导管支架的成型方法 Download PDF

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WO2021068743A1
WO2021068743A1 PCT/CN2020/117010 CN2020117010W WO2021068743A1 WO 2021068743 A1 WO2021068743 A1 WO 2021068743A1 CN 2020117010 W CN2020117010 W CN 2020117010W WO 2021068743 A1 WO2021068743 A1 WO 2021068743A1
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silk fibroin
freeze
solution
mold
drying
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French (fr)
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顾宁
刘鑫
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东南大学
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Priority to US17/602,202 priority Critical patent/US11911536B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/047—Other specific proteins or polypeptides not covered by A61L31/044 - A61L31/046
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C39/00—Shaping by casting, i.e. introducing the moulding material into a mould or between confining surfaces without significant moulding pressure; Apparatus therefor
    • B29C39/22—Component parts, details or accessories; Auxiliary operations
    • B29C39/38—Heating or cooling
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29K—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES B29B, B29C OR B29D, RELATING TO MOULDING MATERIALS OR TO MATERIALS FOR MOULDS, REINFORCEMENTS, FILLERS OR PREFORMED PARTS, e.g. INSERTS
    • B29K2089/00—Use of proteins, e.g. casein, gelatine or derivatives thereof, as moulding material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29L—INDEXING SCHEME ASSOCIATED WITH SUBCLASS B29C, RELATING TO PARTICULAR ARTICLES
    • B29L2031/00—Other particular articles
    • B29L2031/753—Medical equipment; Accessories therefor
    • B29L2031/7542—Catheters

Definitions

  • the invention relates to a forming process of a self-supporting silk fibroin catheter stent, which can be used in the technical field of vascular tissue engineering.
  • vascular rupture injuries caused by natural disasters, violent attacks, traffic accidents and common vascular diseases are extremely common clinically.
  • the rupture of blood vessels not only leads to serious blood supply disorders, but also causes hemorrhagic shock, hematoma, traumatic pain and other collateral diseases, but also brings great heart and life burdens to patients.
  • end-to-end suture surgery can be used directly for non-defect injuries.
  • the main method for the treatment of vascular rupture and defect injuries is blood vessel transplantation.
  • Autologous blood vessel transplantation is the gold standard for the treatment of vascular defect injuries.
  • the source of autologous blood vessels is limited, which will cause two problems. Times trauma.
  • the main clinical solution to this big problem is tissue-engineered vascular grafts.
  • Tissue engineering artificial blood vessels mainly include three elements: seed cells, growth factors and scaffolds.
  • the scaffold mainly provides a microenvironment for the growth of blood vessels and adhesion sites for cell growth, and its role is very important.
  • Silk fibroin is a natural macromolecular protein. Because of its low price, wide sources, good biocompatibility and controllable biodegradability, it is widely used in the field of biomedical materials. The material is mainly composed of 18 kinds of amino acids, and the final degradation products are amino acids and small peptides, which are easily metabolized by the human body and will not cause obvious inflammation and immune rejection. Therefore, silk fibroin is an ideal material choice for vascular stents. In recent years, silk fibroin vascular stents have also been widely developed and applied, and numerous processing methods for silk fibroin vascular stents have also been proposed. At present, the common preparation methods of silk fibroin vascular stents mainly include freeze-drying and electrospinning.
  • the electrospinning technology has been applied well in the preparation of silk fibroin vascular stents.
  • Han Zhichao and others invented the use of electrospinning nanofiber vascular stents (CN102397582A, 2012-04-04); Zhang Yaopeng et al. used electrospinning to prepare A regenerative silk fibroin tissue engineering scaffold containing vascular endothelial growth factor (CN102488929A, 2012-06-13); and Roger et al.
  • the scaffolds prepared by these methods are mostly two-dimensional film scaffolds;
  • Zhu Zhenghua invented the use of freeze-drying to prepare a silk fibroin microporous three-dimensional scaffold (CN102133432A, 2011-07-27).
  • the scaffold is prepared and molded, its moldability is not good. Good, poor tolerance, and the addition of organic solvent hexafluoroisopropanol increases the toxic reaction of the stent.
  • the present invention aims to obtain a three-dimensional porous silk fibroin scaffold with good formability by regulating the preparation of the stent before the freeze-drying process and the optimization of the process during the process, without any additional components.
  • the process is simple to control and the preparation process is simple. It has great application prospects for the preparation of stents by freeze-drying methods in clinics.
  • the purpose of the present invention is to provide a self-supporting silk fibroin catheter stent molding process without any additional components.
  • the method for forming a self-supporting silk fibroin catheter stent of the present invention uses silk fibroin as a raw material to prepare a self-supporting silk fibroin catheter stent by mold casting and freeze-drying molding methods: the specific preparation steps are as follows:
  • the mold includes a three-part structure of a sleeve, an inner core and a cover.
  • the sleeve uses a polyethylene straw with a diameter of 4-6mm, which can effectively prevent the adhesion of the silk fibroin solution.
  • the inner core It is a fiber rod FRP with a diameter of 2-3mm.
  • the cover is designed with stainless steel. One end is closed and the other is not closed. The diameter is consistent with the diameter of the casing.
  • the overall shape and size of the mold are designed according to requirements;
  • Freeze-drying molding After pouring the solution, place it in the refrigerator at -20°C for 6-24 hours. The lyophilizer is pre-cooled and set at -40°C to -35°C, and then the mold after pouring the solution is placed on the pre-cooled layer. The plate is freeze-dried according to the predetermined freeze-drying procedure of the stent.
  • step b The specific preparation steps of the pouring solution described in step b are:
  • step 2 3Put the silk fibroin solution of step 2 in a dialysis bag, dialysis in ddH20 for 3-4 days, then take it out, place it in a container and concentrate to 5-10% in an ultra-clean workbench, and set aside.
  • step c The mold after pouring the solution described in step c is taken out from the refrigerator at -20° C. After removing the mold and transferring it to the lyophilizer, the pre-frozen laminate must be fast, otherwise the solution will easily melt away.
  • the predetermined freeze-drying procedure of the stent in step c follows a strict gradient heating process, the vacuum degree is 300-500 mt, the temperature rises a gradient of 4-6° C., and each temperature gradient is maintained for 100-200 min.
  • the predetermined freeze-drying procedure of the stent in step c needs to be freeze-dried twice.
  • the concentration of 0.5 ⁇ 0.01% (g/ml) NaCO 3 described in step 1 is dissolved with ddH2O.
  • the molecular weight cut-off of the dialysis bag in step 3 is 12000-14000.
  • the dialysate described in step 3 is changed 3-5 times a day.
  • the self-supporting silk fibroin catheter scaffold prepared by the present invention has a three-dimensional porous spatial structure, a large specific surface area, and has both hydrophobic and hydrophilic groups, which is conducive to the growth, adhesion and proliferation of cells, and is the growth of blood vessels. Provide a good microenvironment.
  • the self-supporting catheter stent prepared by the present invention does not use any additional components, retains the good biocompatibility of silk fibroin, and reduces inflammation and immune responses in catheter applications.
  • the self-supporting silk fibroin catheter stent prepared by the present invention has good tolerance and can meet the traction of surgical sutures during the repair of the stent;
  • the self-supporting silk fibroin catheter stent prepared in the present invention has a good slow-release function and can be used as a slow-release carrier for drugs, factors, etc.;
  • the self-supporting silk fibroin catheter scaffold prepared by the present invention can be used not only in vascular tissue engineering, but also in tissue engineering fields such as nerves and skin.
  • Figure 1 is a TBO staining diagram of the self-supporting silk fibroin catheter prepared in Example 2 of the present invention and HUVEC co-cultured with vascular endothelial cells.
  • Fig. 2 is a graph showing the viability of CCK-8 cells co-cultured with the self-supporting silk fibroin catheter prepared in Example 3 of the present invention and HUVEC of vascular endothelial cells.
  • Figure 3 is a graph showing the hemolysis test of a self-supporting silk fibroin catheter prepared in Example 4 of the present invention.
  • the present invention provides a self-supporting silk fibroin catheter stent forming process without any additional components, including the following steps:
  • step (3) The silk fibroin solution described in step (2) needs to be dialyzed through a dialysis bag to obtain the dialysis silk fibroin solution:
  • step (3) The silk fibroin solution after dialysis described in step (3) is concentrated in a fume hood, filtered, and set aside.
  • step (4) The concentrated silk fibroin solution described in step (4) is injected into the catheter mold;
  • the lyophilizer is pre-frozen and turned on until the temperature of the shelf reaches -40 to -35°C;
  • step(6) The pre-frozen mold support and take out the cover and inner core of the two ends of the mold and place them in step(7)Pre-frozen laminate;
  • the natural silk mentioned in step (1) is mulberry silk, which is mainly composed of silk fibroin and sericin.
  • the ratio of the two components is about 17:3-4:1.
  • Silk fibroin is the main component of silk.
  • Amino acid composition of which serine (Ser).
  • Alanine (Ala) and glycine (Gly) account for about 85% of the total composition, and the molar ratio of the three is 1:3:4;
  • the described sodium carbonate solution desericin treatment method is through 0.5 ⁇ 0.01% sodium carbonate Alkaline solution treatment, ddH 2 0 washing after 2-3 times of boiling.
  • the ternary solution described in step (2) is composed of anhydrous calcium chloride, anhydrous ethanol and double distilled water, the molar ratio of the three is 1:2:8, the dissolution temperature is 70-75°C, and the dissolution time is when the solution is After all is dissolved, continue to heat and stir at this temperature for 30-40 minutes; the volume ratio of silk fibroin to solution is 1:4-6.
  • the molecular weight cut-off of the dialysis bag described in step (3) is 12000-14000
  • the dialysis solution is ddH 2 0
  • the dialysis time is 3-4 days
  • the ddH 2 0 is changed 3-5 times a day.
  • the fume hood described in step (4) must be clean, the concentration of the solution is 5-10%, and the filtration adopts a 50-100 ⁇ m nylon filter, and it is placed in a refrigerator at 4°C until it is used.
  • the catheter mold described in step (5) mainly includes a three-part structure of a sleeve, an inner core and a cover.
  • the sleeve uses a polyethylene straw with a diameter of 4-6mm, which can effectively prevent the adhesion of the silk fibroin solution.
  • the inner core It is a fiber rod FRP with a diameter of 2-3mm.
  • the cover is made of stainless steel. One end is closed and the other is not closed. The diameter is consistent with the diameter of the casing.
  • the overall shape and size of the mold can be designed according to requirements.
  • the pre-freezing time described in step (6) can also be appropriately extended.
  • the lyophilizer layer board described in step (7) is pre-cooled for more than 120 minutes.
  • the cover at both ends of the mold described in step (8) and the inner core must be taken out quickly to prevent the silk fibroin solution from melting, and the bracket must be placed in direct contact with the plate, and continue to pre-freeze for more than 40-60 minutes.
  • the predetermined freeze-drying procedure of the stent described in step (9) follows a strict gradient heating process.
  • the freezing process includes four stages: a pre-freezing stage, a freezing vacuum transition stage, a gradient heating freeze-drying stage and a secondary freeze-drying stage.
  • Pre-freezing stage maintain the pre-freezing temperature from -40°C to -35°C for 300-500min; freezing vacuum transition stage: keep the freezing temperature consistent with the pre-freezing stage and maintain 100-200min; gradient heating freeze-drying stage: start temperature and The freeze-vacuum transition stage is consistent, and each temperature gradient is maintained for 100-200min; the temperature is increased by 4-6°C and a gradient; the second freeze-drying stage: the temperature is higher than the maximum temperature of the gradient temperature-raising freeze-drying stage by 5-10°C, and the maintenance time reaches Above 800min; the vacuum degree of the whole process is 300-500mT, and keep the same value.
  • Step 1 Preparation of silk fibroin (desicin)
  • Step 2 Preparation of silk fibroin solution
  • Example 2 Weigh 20g of the silk fibroin solid prepared in Example 1 and place it in a fresh-keeping bag for use. Prepare a 250ml beaker, then weigh 37g of anhydrous calcium chloride and place it in the beaker, add 48ml of double distilled water, anhydrous 40ml of ethanol to fully dissolve, put the dissolved solution in a magnetic stirrer and stir evenly. Cover the beaker with a cling film to prevent the ethanol from evaporating. The heating temperature is maintained at 72°C, and 20g of silk fibroin solid is kept under stirring.
  • Step 3 Mold design and material selection
  • the main components of the mold include a three-part structure of sleeve, inner core and cover.
  • the sleeve uses a 6mm straw with a length of 80mm, which effectively prevents the silk fibroin solution from sticking to the sleeve wall after freeze-drying and cannot be taken out.
  • the core is a fiber rod FRP with a diameter of 2mm, which makes the stent form a tubular structure and is not easy to adhere to the silk fibroin.
  • the two ends are covered with stainless steel, and one end is closed. This end is wide and the end is 8mm in diameter and 5mm in height.
  • the role of the narrow end is to fix the inner core, with an inner diameter of 2.5mm, an outer diameter of 4.5mm, and a height of 6mm; the other end has a small hole at the wide end, the inner diameter of the hole is 2.5ml, the outer diameter is 7mm, and the small hole and the narrow end fix the inner diameter of the inner core Keep the same, the narrow end outer diameter is 4.5mm.
  • a sealing film is required to seal the closed end.
  • Step 4 Preparation of self-supporting silk fibroin catheter scaffold
  • the program is divided into four stages: pre-freezing stage, freezing vacuum transition stage, gradient heating freeze-drying stage and secondary Freeze-drying stage.
  • the pre-freezing stage program is: freezing temperature -40°C, holding time 300min, vacuum degree 475T; freezing vacuum transition stage: freezing temperature -40°C, holding time 100min, vacuum degree 300mT; Gradient heating freeze-drying stage: program operation see Table 1 ; Second freeze-drying stage: temperature 25°C, maintenance time 1250min, vacuum degree 500mT, just take out the sample after running.
  • Table 1 is a program table of the gradient heating stage of the self-supporting silk fibroin vascular stent prepared in Example 1 of the invention.
  • Example 2 Co-cultivation of silk fibroin catheter scaffold and HUVEC for observation of proliferation morphology
  • the silk fibroin catheter prepared in Example 1 was first cross-linked with absolute ethanol to form a water-insoluble structure, and then the silk fibroin catheter was cut to a length of about 5mm, and then the catheter was prepared into a freezing table for frozen sectioning, and the section collection device Use PLL-coated small round glass slides for 24-well culture plates with a thickness of about 30 ⁇ m, and then place the glass slides containing silk fibroin on the 24-well culture plate on the ultra-clean workbench. Sterilize with UV overnight, then add 1.5ml of 75% ethanol to each hole for 30-40min, and then wash with the same amount of 0.01M PBS at least 3 times, 20min each time, dry naturally and leave it for cell inoculation.
  • HUVEC HUVEC
  • vascular endothelial cells set up a blank control for duct sections without silk fibroin. Inoculate HUVEC cells on the material. The number of cells inoculated in each well is 5 ⁇ 10 4 cells. 500 ⁇ L of cell complete medium (complete medium configuration: 45ml DMEM+5ml FBS+500 ⁇ L double antibody) is incubated at 37°C with 5% CO 2 Box incubation. Take it out at the scheduled time point 1, 3, and 5 days.
  • the removed material was gently washed twice with 0.01M PBS for about 5 minutes each time, fixed with 4% paraformaldehyde for 40 minutes, and then washed twice with 0.01M PBS for about 10 minutes each time, and TBO dye solution was added after washing ( Adopted by sigma company, 0.5g plus PBS 200ml when in use), the dye solution can cover the material completely, and dye for 15min at room temperature. After staining, wash with 0.01M PBS for several times until the slides appear light blue. Observation of staining under light microscope is shown in Figure 1. (The left column is a blank control group without silk fibroin, and the right column is a catheter containing silk fibroin.
  • the upper, middle and lower ones are the cell proliferation at different time points on day 1, 3, and 5 respectively).
  • the results showed that the HUVEC proliferation in the experimental group and the control group was good at different time points, without obvious differences, and the cell morphology was good.
  • the silk fibroin catheter scaffold has good biocompatibility, which is conducive to cell adhesion and growth. .
  • Example 3 Co-cultivation of silk fibroin catheter scaffold and HUVEC for cell viability observation
  • the silk fibroin catheter scaffold prepared in Example 1 was cross-linked with absolute ethanol to form a water-insoluble structure.
  • the catheter was cut to a length of 10 mm and then placed in a 24-well culture plate. A total of five parallel samples were set in each group. The hole without silk fibroin catheter was used as a blank control. Place the 24-well culture plate containing the sample on the ultra-clean workbench, sterilize it overnight, add 1.5ml of 75% ethanol to each well for 30-40 minutes, wash it with the same amount of 0.01M PBS 5 times, and dry it naturally Leave it for cell seeding.
  • the cells are HUVEC, which is vascular endothelial cell.
  • the silk fibroin catheter scaffold prepared in Example 1 was first cross-linked with absolute ethanol to form a water-insoluble structure, and weighed 50 mg for use.
  • the blood collection method is heart blood collection.
  • anesthetize the big-eared white rabbit with 3% sodium pentobarbital (1.2ml/kg) fix the big-eared white rabbit supine on the operating table, shave the heart part and sterilize it with iodophor, choose a heartbeat
  • the most obvious part of the beating is punctured, and the needle is pierced into the heart to obtain an appropriate amount of blood and then pulled out quickly, and then the hemolytic test is performed.
  • hemolysis% (OD SF- OD negative ) / (OD positive- OD negative ) ⁇ 100%
  • the results of the study are as follows
  • the hemolysis rate of the silk fibroin catheter stent is 0.399%, which is much lower than the national standard of 5%. This result shows that the silk fibroin catheter stent has no obvious hemolysis, which means that the silk fibroin catheter stent has Good blood compatibility.
  • Table 2 is the experimental test data table of hemolysis of the self-supporting silk fibroin vascular stent prepared in Example 1 of the present invention.

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Abstract

一种自支撑丝素蛋白导管支架的成型方法，以丝素蛋白为原料，通过模具浇注和冷冻干燥成型的工艺制备得到所述的导管支架。所述的原料是从天然桑蚕丝中提取的丝素蛋白，所述的模具是中空管状模具，模具外壳是直径6mm聚乙烯透明吸管，内芯是直径3mm纤维棒FRP，两端封口。所述的冷冻干燥工艺包括以下步骤：浇注；预冷冻；去除模具放入预冷冻的冻干层板；冷冻干燥；冷冻干燥过程包括：⑴预冷冻；⑵冷冻真空过渡阶段：⑶梯度升温冻干阶段：⑷二次冻干阶段，针对冻干支架的规格，严格调控冷冻干燥阶段。所制备的支架成型良好，耐受性好，不需要任何添加成份，支架呈现三维多孔空间结构，工艺简单，可满足临床组织工程血管支架的使用要求。

Description

一种自支撑丝素蛋白导管支架的成型方法 技术领域
本发明涉及一种自支撑丝素蛋白导管支架的成型工艺，可用于血管组织工程技术领域。
背景技术
由于自然灾害、暴力侵袭、交通事故以及常见的血管疾病导致等导致的血管断裂损伤在临床上极为常见。血管断裂损伤不仅导致严重的供血障碍，引起包括失血性休克、血肿以及创伤性疼痛和其它连带疾病，还会给患者带来极大的心里和生活负担。一般对于无缺损损伤直接采用端对端缝合手术即可，但对于血管断裂缺损损伤的治疗主要方法是血管移植，自体血管移植是治疗血管缺损损伤的金标准，但是自体血管来源有限，会造成二次创伤。临床解决这一大难题的主要办法就是组织工程血管移植物，因此临床上研究主要集中在人工血管作为替代物。组织工程人工血管主要包括三点要素：种子细胞、生长因子和支架。其中支架在里面主要为血管生长提供一个微环境和细胞生长的粘附位点，其作用举足轻重。
丝素蛋白是一种天然的大分子蛋白质，因其价格底低廉，来源广泛，且具有良好的生物相容性和可控的生物降解性而被广泛的应用到生物医用材料领域。材料主要有18种氨基酸组成，最终降解产物为氨基酸和小肽，易被人体代谢，不会引起明显的炎症反应和免疫排斥。因此丝素蛋白是血管支架理想的材料选择。近年来，丝素蛋白血管支架也被广泛的开发应用，众多的丝素蛋白血管支架的工艺方法也被提出来。目前，常见的丝素蛋白血管支架制备方法主要有冷冻干燥、静电纺丝等。目前静电纺丝技术在制备丝素蛋白血管支架中已经应用良好，例如：韩志超等发明了利用静电纺丝法纳米纤维血管支架(CN102397582A,2012-04-04)；张耀鹏等人利用静电纺丝制备出含血管内皮生长因子的再生丝素蛋白组织工程支架(CN102488929A，2012-06-13)；以及罗杰等人利用溶喷纺丝制备了丝素蛋白组织工程支架(CN109667059A，2019-04-23)，这些方法制备出的支架多为二维薄膜支架；朱正华发明了利用冷冻干燥制备一种丝素蛋白微孔三维支架(CN102133432A，2011-07-27)，虽然支架制备成型，但成型性不好，耐受性差同时还添加了有机溶剂六氟异丙醇增加了支架的毒性反应。目前在利用冷冻干燥法制备血管支架成型还很困难，支架成型差，耐受性差，无法满足临床使用需求。
本发明旨在通过调控支架在冷冻干燥过程前制备和过程中的工艺调控优化，不需要任何添加成份，即可获得成型性良好的三维多孔丝素蛋白支架，此工艺调控简单，制备过程简单，对临床采用冻干方法制备支架具有重大的应用前景。
发明内容
技术问题：针对现有的冻干技术成型性不好，耐受性差不足，本发明的目的是提供一种自支撑丝素蛋白导管支架成型工艺且不需要任何添加成份。
技术方案：本发明的一种自支撑丝素蛋白导管支架的成型方法，采用丝素蛋白为原料，通过模具浇注和冷冻干燥成型的方法制备出自支撑丝素蛋白导管支架：具体的制备步骤如下：
a.模具结构的设计：该模具包括套管、内芯和封盖三部分结构，套管采用的是聚乙烯吸管，直径4-6mm，可以有效地防止丝素蛋白溶液的粘附，内芯是直径2-3mm纤维棒FRP，封盖采用不锈钢设计，一端密闭一端不密闭，直径大小和套管直径吻合，模具整体形状和大小按照需求设计；
b.浇注溶液的制备：天然桑蚕丝首先需经过碳酸钠溶液脱丝胶处理，然后采用三元溶液CaCl 2-EtOH-H 2O进行溶解后透析、浓缩、待用；
c.冷冻干燥成型：浇注溶液后先放置于-20度冰箱冷冻6-24h，冻干机预先制冷置-40℃到-35℃，然后将浇注溶液后的模具放置于预先制冷的好的层板，按照支架预定的冷冻干燥程序冷冻干燥。
其中，
步骤b所述的浇注溶液的具体制备步骤为：
①称取天然桑蚕丝加入到浓度为0.5±0.01％(g/ml)的NaCO 3溶液中，加热到95-100℃，从煮沸开始计时加热30-40min，然后用ddH 20洗涤去除溶解下来的丝胶，重复上述步骤2-3次，得到丝素蛋白，晾干，待用；
②称取步骤①丝素蛋白溶解于三元溶液中CaCl 2/EtOH/H 2O＝1：2：8；三元溶液的配置：无水CaCl 2先溶于ddH2O中，再加入无水乙醇，将丝素蛋白分批次缓慢加入三元溶液中并置于磁力搅拌器中搅拌加热至72-75℃，完全溶解后继续搅拌20-30min，得丝素蛋白溶液；
③将步骤②的丝素蛋白溶液置于透析袋中于ddH20透析3-4天后取出，置于容器中于超净工作台中浓缩至5-10％，待用。
所述步骤c所述的浇注溶液后的模具从-20℃冰箱取出后去除模具转移至冻干机预冷冻层板一定要快，否则溶液容易融化掉。
所述步骤c所述的支架预定的冷冻干燥程序遵循严格的梯度升温过程，真空度300-500mt，温度升温4-6℃一个梯度，每个温度梯度维持时间100-200min。
所述步骤c所述的支架预定的冷冻干燥程序要进行二次冻干。
步骤①所述的浓度0.5±0.01％(g/ml)NaCO 3采用ddH2O溶解。
步骤③所述的透析袋截留分子量为12000-14000。
步骤③所述的透析液每天更换3-5次。
有益效果：
1、本发明制备的自支撑丝素蛋白导管支架具有呈现三维多孔的空间结构，具有大的比表面积，同时具备疏水和亲水基团，有利于细胞的生长、粘附和增殖，为血管生长提供良好的微环境。
2、本发明制备的自支撑导管支架未使用任何添加成份，保留了丝素蛋白良好的生物相容性，减少导管应用的炎症反应和免疫反应。
3、本发明制备的自支撑丝素蛋白导管支架有较好的耐受性，可以满足支架在修复中手术缝合线的牵拉；
4、本发明制备的自支撑丝素蛋白导管支架具有良好的缓释功能，可用作药物、因子等缓释载体；
5、本发明制备的自支撑丝素蛋白导管支架不仅可以用作血管组织工程，也可以用作神经、皮肤等组织工程领域。
附图说明
图1为本发明实施例2制备的自支撑丝素蛋白导管与血管内皮细胞HUVEC共培养TBO染色图。
图2为本发明实施例3制备的自支撑丝素蛋白导管与血管内皮细胞HUVEC共培养CCK-8细胞活力检测图。
图3为本发明实施例4制备的自支撑丝素蛋白导管溶血性测试图。
具体实施方式
本发明提供了一种自支撑丝素蛋白导管支架成型工艺且不需要任何添加成份，包括以下步骤：
⑴将天然蚕丝用碳酸钠溶液进行脱丝胶处理获得丝素蛋白；
⑵步骤⑴所述的丝素蛋白采用三元溶液进行溶解处理获得丝素蛋白溶液；
⑶步骤⑵所述的丝素蛋白溶液需经过透析袋透析获得透析后丝素蛋白溶液：
⑷步骤⑶所述的透析后丝素蛋白溶液置于通风橱内浓缩，过滤，待用。
⑸步骤⑷所述的浓缩丝素蛋白溶液注入导管模具；
⑹密封置于-20℃冰箱进行预冷冻6-24h；
⑺冻干机预冷冻打开直至层板温度达到-40到-35℃；
⑻取出步骤⑹预冷冻的模具支架并取出模具的两端的封盖和内芯置于步骤⑺预冷冻层板；
⑼设定冷冻干燥循环程序对步骤⑻所述的支架进行冷冻干燥；
其中步骤⑴所述的天然蚕丝是桑蚕丝，主要有丝素蛋白和丝胶蛋白构成，两者的成份质 量比约为17:3-4:1，丝素蛋白是蚕丝的主要成分由18种氨基酸构成，其中丝氨酸(Ser)。丙氨酸(Ala)和甘氨酸(Gly)约占总成份的85％，三者摩尔比为1:3:4；所述的碳酸钠溶液脱丝胶处理方法是，通过0.5±0.01％碳酸钠碱性溶液处理，经过2-3次煮沸后ddH 20洗涤。
其中步骤⑵所述的三元溶液由无水氯化钙、无水乙醇和双蒸水组成，三者的摩尔比为1:2：8，溶解温度为70-75℃，溶解时间为当溶液全部溶解后继续在此温度下加热搅拌30-40min；丝素蛋白与溶液体积比1:4-6。
其中步骤⑶所述的透析袋截留分子量为12000-14000，透析溶液是ddH 20,透析时间为3-4天，每天更换ddH 20 3-5次。
其中步骤⑷所述的通风橱必须洁净，溶液浓缩浓度为5-10％，过滤采用50-100μm尼龙过滤网，待用放置于4℃冰箱。
其中步骤⑸所述的导管模具主要包括套管、内芯和封盖三部分结构，套管采用的是聚乙烯吸管，直径4-6mm，可以有效地防止丝素蛋白溶液的粘附，内芯是直径2-3mm纤维棒FRP，封盖采用不锈钢设计，一端密闭一端不密闭，直径大小和套管直径大下吻合，模具整体形状和大小可按照需求设计。
其中步骤⑹所述的的预冷冻时间也可以适当延长。
其中步骤⑺所述的冻干机层板预冷120min以上。
其中步骤⑻所述的模具两端封盖和内芯取出必须要快，防止丝素溶液融化，支架置于层板必须与板直接接触，继续预冷冻40-60min以上。
步骤⑼所述的支架预定的冷冻干燥程序遵循严格的梯度升温过程，冷冻过程包括四个阶段：预冷冻阶段、冷冻真空过渡阶段、梯度升温冻干阶段和二次冻干阶段。预冷冻阶段：维持预冷冻温度-40℃到-35℃，维持时间300-500min；冷冻真空过渡阶段：冷冻温度与预冷冻阶段保持一致，维持100-200min；梯度升温冻干阶段：开始温度与冷冻真空过渡阶段保持一致，每个温度梯度维持时间100—200min；温度升温4-6℃一个梯度；二次冻干阶段：温度高于梯度升温冻干阶段最高温度5-10℃，维持时间达到800min以上；整个过程真空度为300-500mT,并保持数值一致。
为了加深对发明的理解，下面结合实施例对本发明做进一步详述。
实施例1：
步骤1：丝素蛋白的的制备(脱丝胶)
称取购买的桑蚕丝(包有丝胶)40-50g到不锈钢锅中，然后称取无水碳酸钠固体颗粒充分溶解到2L的双蒸水中，将溶解好的碳酸钠(浓度0.5％)溶液倒入含有桑蚕丝的不锈钢锅中，用玻璃棒充分按压使桑蚕丝完全浸没于碳酸钠溶液中，然后将锅置于电磁炉上加热直至煮沸，从煮沸时开始计时，约30min后取出，为了受热均匀，蒸煮期间应用玻璃棒多次搅拌， 然后将煮好的桑蚕丝用双蒸水洗涤3-4次，此时丝胶被清除，丝素蛋白裸露。为了使丝胶被充分清除，需要重复上述步骤2次，然后将煮好的丝素蛋白用双蒸水洗涤数次，直至PH试纸检测呈中性，然后脱水，置于通风处晾干，待用。
步骤2：丝素蛋白溶液的制备
称取实施例1中制备的丝素蛋白固体20g置于保鲜袋中待用，准备一个250ml的烧杯，然后称取无水氯化钙37g放于烧杯中，，加入双蒸水48ml，无水乙醇40ml使其充分溶解，将溶解后的溶液放入磁力搅拌器中搅拌均匀，烧杯上面覆盖一层保鲜膜以防止乙醇挥发，加热温度维持在72℃，在搅拌条件下将20g丝素蛋白固体分多次加入，加完继续搅拌20-30min使丝素蛋白充分溶解，然后取出室温下放凉，最后放入透析袋(截留分子量：12000-14000)透析3天，每天更换透析液(ddH 2O)4次。最后取出放入通风橱浓缩至8％，放置4℃冰箱保存、待用。
步骤3：模具的设计和材料的选择
模具的主要组成部分括套管、内芯和封盖三部分结构，套管采用的是6mm吸管，长度80mm，有效地防止丝素蛋白溶液在冻干后粘附在套管壁无法取出的缺陷。芯是选择直径2mm的纤维棒FRP,其作用使支架形成管状结构并不易粘附在丝素蛋白上，两端采用不锈钢封盖，一端是封闭的，此端宽头端直径8mm，高度5mm，窄端作用是为了固定内芯，内径2.5mm，外径4.5mm，高度6mm；另一端宽头端留有小孔，小孔内径2.5ml，外径7mm，小孔与窄端固定内芯内径保持一致，窄端外径4.5mm。在注入溶液时为了防止封闭端溶液渗漏需要封口膜将封闭端封住。
步骤4：自支撑丝素蛋白导管支架的制备
将1.5ml的实施例2制备的丝素蛋白溶液注入实施例3设计的模具中，然后将其放置15ml的离心管中置于试管架中于-20℃冰箱预冷冻12h，然后将冻干机冷冻打开降温直至层板温度达到-40℃，然后将丝素蛋白冷冻液取出以最快的速度去除模具的两端和内芯且丝素蛋白不呈现流动相，然后将其放到预冷冻温度-40℃的层板上维持时间100min，然后关闭制冷，打开预先设定好的自动冻干程序，程序分为四个阶段：预冷冻阶段、冷冻真空过渡阶段、梯度升温冻干阶段和二次冻干阶段。预冷冻阶段程序为：冷冻温度-40℃，维持时间300min，真空度475T；冷冻真空过渡阶段：冷冻温度-40℃，维持时间100min，真空度300mT；梯度升温冻干阶段：程序运行见表1；二次冻干阶段：温度25℃，维持时间1250min，真空度500mT,运行结束后取出样品即可。
表1为本发明实施例1制备的自支撑丝素蛋白血管支架梯度升温阶段程序表。
程序号 温度(℃) 时间(min) 真空度(mT)
1 -40 200 300mT
2 -35 200 300mT
3 -30 200 300mT
4 -25 200 300mT
5 -20 200 300mT
6 -15 200 300mT
7 -10 200 300mT
8 -5 200 300mT
9 0 200 300mT
10 5 200 300mT
11 10 200 300mT
12 15 200 300mT
13 20 200 300mT
表1
实施例2：丝素蛋白导管支架与HUVEC共培养进行增殖形态观察
将实施例1制备的丝素蛋白导管，先经过无水乙醇交联后形成水不溶性结构，然后将丝素蛋白导管裁剪约5mm长度，然后将导管制备成冻台进行冰冻切片，切片的收集装置采用经过PLL包被的用于24孔培养板的小圆玻片，切片的厚度约为30μm，然后将含有丝素蛋白的玻片置于24孔培养板放于超净工作台。紫外灭菌过夜，然后每孔加入1.5ml 75％的乙醇消毒30-40min后用等量的0.01M PBS洗涤至少3次，每次20min，自然晾干后留待细胞接种，细胞选用血管内皮细胞HUVEC并设置无丝素蛋白导管切片的空白对照。将HUVEC细胞接种到材料上，每个孔接种细胞数量5×10 4个，细胞完全培养基500μL(完全培养基配置：45ml DMEM+5ml FBS+500μL双抗)置于37℃5％CO 2培养箱孵育。在预定的时间点1、3、5天取出。将取出后的材料用0.01M PBS轻轻洗涤2次，每次约5min，4％多聚甲醛固定40min，然后再用0.01M PBS洗涤2次，每次约10min，洗涤后加TBO染液(sigma公司采用，使用时0.5g加PBS 200ml配置用)，染液全覆盖材料即可，室温下染色15min。染色后用0.01M PBS洗涤数次直至玻片呈现淡蓝色，光镜下观察染色情况如图1所示，(左栏为没有丝素蛋白的空白对照组，右栏为含有丝素蛋白导管切片的实验组，上中下分别为时间点1、3、5天不同时间点细胞增殖情况)。结果表明，在不同的时间点细胞实验组和对照组HUVEC增殖情况良好，未表现出明显的差异，细胞形态良好，丝素蛋白导管支架具有良好的生物相容性，利于细胞 的粘附和生长。
实施例3：丝素蛋白导管支架与HUVEC共培养进行细胞活力观察
将实施例1制备的丝素蛋白导管支架，先经过无水乙醇交联后形成水不溶性结构，将导管裁剪成10mm长度，然后放入24孔培养板内，每组共设置五个平行样，无丝素蛋白导管的孔作为空白对照。将装有样品的24孔培养板置于超净工作台，紫外灭菌过夜，然后每孔加入1.5ml75％的乙醇消毒30-40min后用等量的0.01M PBS洗涤5次，自然晾干后留待细胞接种。细胞选用血管内皮细胞HUVEC。首先，向每个孔中加入1ml DMEM溶液，并在含有5％CO  2的培养箱中于37℃温育24小时。培养期结束后，在DMEM溶液中加入10％FBS和抗生素(100U/ml青霉素和100μg/ml链霉素)，得到完全培养基。然后，将HUVEC细胞接种到导管上，细胞密度为1×10 4个细胞/ml。最后，将培养板在37℃，5％CO  2环境下孵育1天和2天后，除去培养基，用PBS(0.01M)冲洗非贴壁细胞三次。随后，将新鲜培养基和CCK-8试剂(10：1)的混合物加入样品中，并在标准培养条件下于37℃温育4小时。然后将悬浮液(200μL/孔)移入96孔板中，用酶标仪在450nm处测量吸光度。结果如图2所示，结果表明对照组合和丝素蛋白导管组相比，1天的时候两者的增殖活力相当未表现出明显的差异，2天的时候丝素蛋白导管组增殖活力超过了对照组，这表明丝素蛋白导管具有良好的细胞相容性，且可以促进细胞增殖的能力，丝素蛋白三维空间结构为细胞生长繁殖提供了良好的微环境，具有促进血管损伤修复的能力。
实施例4：丝素蛋白导管支架血液相容性检测
将实施例1制备的丝素蛋白导管支架，先经过无水乙醇交联后形成水不溶性结构，称重50mg备用。选择健康的成年大耳白兔(约2.5kg/只)用于采血，采血方法为心脏取血。首先，将大耳白兔用3％的戊巴比妥钠麻醉(1.2ml/kg)后，将大耳白兔仰卧固定在手术台上，心脏部位剃毛并用碘伏消毒处理，选择心博跳动最明显部位穿刺，针头刺入心脏取得适量的血液后迅速拔出，然后进行溶血性实验。将采集的血液加入抗凝血管，加生理盐水1；1稀释待用。取备好的50mg样品方法1.5ml离心管，加入生理盐水1ml于30℃放置30min，另取蒸馏水1ml作阳性对照，生理盐水1ml作阴性对照于同样条件下放置30min，时间到后每个离心管加入100μL生理盐水稀释后的兔血，37℃放置3h。然后以8000rmp速度离心5min，取上清液用酶标仪测545nm处OD值，溶血计算公式：溶血％＝(OD SF-OD 阴性)/(OD 阳性-OD 阴性)×100％，研究结果如图3及表2所示，丝素蛋白导管支架的溶血率为0.399％，数值远低于国家标准5％，该结果表明丝素蛋白导管支架无明显溶血，也就是说丝素蛋白导管支架具有良好的血液相容性。
表2为本发明实施例1制备的自支撑丝素蛋白血管支架溶血性实验测试数据表。
Figure PCTCN2020117010-appb-000001
表2

Claims (8)

  1. 一种自支撑丝素蛋白导管支架的成型方法，其特征在于，采用丝素蛋白为原料，通过模具浇注和冷冻干燥成型的方法制备出自支撑丝素蛋白导管支架：具体的制备步骤如下：
    a.模具结构的设计：该模具包括套管、内芯和封盖三部分结构，套管采用的是聚乙烯吸管，直径4-6mm，可以有效地防止丝素蛋白溶液的粘附，内芯是直径2-3mm纤维棒FRP，封盖采用不锈钢设计，一端密闭一端不密闭，直径大小和套管直径吻合，模具整体形状和大小按照需求设计；
    b.浇注溶液的制备：天然桑蚕丝首先需经过碳酸钠溶液脱丝胶处理，然后采用三元溶液CaCl 2-EtOH-H 2O进行溶解后透析、浓缩、待用；
    c.冷冻干燥成型：浇注溶液后先放置于-20度冰箱冷冻6-24h，冻干机预先制冷置-40℃到-35℃，然后将浇注溶液后的模具放置于预先制冷的好的层板，按照支架预定的冷冻干燥程序冷冻干燥。
  2. 根据权利要求1所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，步骤b所述的浇注溶液的具体制备步骤为：
    ①称取天然桑蚕丝加入到浓度为0.5±0.01％(g/ml)的NaCO 3溶液中，加热到95-100℃，从煮沸开始计时加热30-40min，然后用ddH 20洗涤去除溶解下来的丝胶，重复上述步骤2-3次，得到丝素蛋白，晾干，待用；
    ②称取步骤①丝素蛋白溶解于三元溶液中CaCl 2/EtOH/H 2O＝1：2：8；三元溶液的配置：无水CaCl 2先溶于ddH2O中，再加入无水乙醇，将丝素蛋白分批次缓慢加入三元溶液中并置于磁力搅拌器中搅拌加热至72-75℃，完全溶解后继续搅拌20-30min，得丝素蛋白溶液；
    ③将步骤②的丝素蛋白溶液置于透析袋中于ddH20透析3-4天后取出，置于容器中于超净工作台中浓缩至5-10％，待用。
  3. 根据权利要求1所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，所述步骤c所述的浇注溶液后的模具从-20℃冰箱取出后去除模具转移至冻干机预冷冻层板一定要快，否则溶液容易融化掉。
  4. 根据权利要求1所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，所述步骤c所述的支架预定的冷冻干燥程序遵循严格的梯度升温过程，真空度300-500mt，温度升温4-6℃一个梯度，每个温度梯度维持时间100-200min。
  5. 根据权利要求1所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，所述步骤c所述的支架预定的冷冻干燥程序要进行二次冻干。
  6. 根据权利2所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，步骤①所述的浓度0.5±0.01％(g/ml)NaCO 3采用ddH2O溶解。
  7. 根据权利2所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，步骤③所述的透析袋截留分子量为12000-14000。
  8. 根据权利2所述的一种自支撑丝素蛋白导管支架的成型方法，其特征在于，步骤③所述的透析液每天更换3-5次。
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