CN111714704A

CN111714704A - PGS/SF electrospun artificial blood vessel and preparation method thereof

Info

Publication number: CN111714704A
Application number: CN202010518963.6A
Authority: CN
Inventors: 赵荟菁; 孟凯; 张迎梅
Original assignee: Suzhou University; Nantong Textile and Silk Industrial Technology Research Institute
Current assignee: Suzhou University; Nantong Textile and Silk Industrial Technology Research Institute
Priority date: 2020-06-09
Filing date: 2020-06-09
Publication date: 2020-09-29
Anticipated expiration: 2040-06-09
Also published as: CN111714704B

Abstract

The invention relates to a PGS/SF electrospun artificial blood vessel and a preparation method thereof. The preparation method of the PGS/SF electrospun artificial blood vessel comprises the following steps: dissolving the PGS prepolymer and SF in an organic solvent to obtain a spinning solution, carrying out electrostatic spinning on the spinning solution, receiving the micro-nano-scale fibers by adopting a cylindrical receiving device rotating around the axis of the receiving device, winding the micro-nano-scale fibers outside the receiving device to form a tubular PGS/SF electrospun membrane, and drying and curing the tubular PGS/SF electrospun membrane after the spinning is completed to obtain the PGS/SF electrospun artificial blood vessel. The invention utilizes SF to improve the spinnability and the fibroblasticity of PGS, and successfully obtains PGS/SF electrospun artificial blood vessel, the caliber of the electrospun artificial blood vessel is less than 6mm, and the electrospun artificial blood vessel has the excellent elasticity of PGS and the excellent biological and mechanical properties of SF.

Description

PGS/SF electrospun artificial blood vessel and preparation method thereof

Technical Field

The invention relates to an electrospun membrane, in particular to a PGS/SF electrospun artificial blood vessel and a preparation method thereof.

Background

Vascular diseases are one of the main diseases causing high human mortality, and for patients with serious injured parts, surgical operations such as vascular graft replacement or bypass are usually adopted, so that the vascular diseases are an effective treatment means. Currently, large and medium-caliber artificial blood vessels (>6mm) are used clinically, such as artificial blood vessels made of Dacron (Dacron) and expanded polytetrafluoroethylene (ePTFE) by a weaving or braiding method. Small-caliber artificial blood vessels (<6mm) are still in the research stage, and because the small-caliber artificial blood vessels are not matched with the compliance of host blood vessels after being transplanted, stress concentration areas are formed at anastomotic sites, so that the problems of thrombus, intimal hyperplasia at the anastomotic sites, aneurysm and the like occur, and the small-caliber artificial blood vessels are the main reasons of transplantation failure.

Vessel compliance refers to the ability of the vessel wall to expand and contract when subjected to the radially pulsating pressure of blood flow. The compliance of the artificial blood vessel is directly related to the elasticity of the material used, and generally, the better the elasticity of the material, the better the compliance of the artificial blood vessel. Therefore, it is necessary to select a biomaterial with good elasticity to prepare a small-caliber artificial blood vessel with good compliance.

The modulus of the biological elastomer is matched with most of soft tissues and organs of a human body, so that the biological elastomer can be used for diagnosing, repairing or replacing the soft tissues of the human body. Polysebacylic acid glyceride (PGS) is a biodegradable non-linear three-dimensional network thermosetting polyester elastomer, is easy to synthesize, has elasticity, biocompatibility and biodegradability, and is a typical biological elastomer. Based on good performance of PGS, the PGS is mainly applied to soft tissue replacement and soft tissue engineering, such as cardiac muscle, blood vessels, nerves, cartilage, retina and tympanic membrane, and is also useful for research of drug delivery carriers and tissue adhesion materials.

The electrostatic spinning is simple in operation and low in cost, is an effective way for obtaining superfine fibers, can simulate in-vivo extracellular matrix to the maximum extent, promotes cell adhesion, growth and proliferation, and provides a better environment for tissue regeneration. The preparation of PGS by the electrostatic spinning method has certain difficulty, and the solidified and crosslinked PGS is insoluble and not molten, cannot be used for preparing electrostatic spinning and cannot find a proper solvent for dissolution, so that the PGS can be processed only at a prepolymer stage. The PGS prepolymer has low molecular weight, cannot be formed by electrostatic spinning, and needs to be assisted by a material with good fiber forming property to form the micro-nanofiber.

CN 107693846A discloses a preparation method of a bionic vascularized soft tissue with a multilayer vascular structure, wherein the bionic vascularized soft tissue contains a vascular channel-like structure, the structure is made of at least one of polycaprolactone, polylactic acid, polyglycolic acid, poly-glycerol sebacate and polylactide-glycolide copolymer, the vascular channel-like structure is prepared by a coating method, and the vascular channel-like structure does not contain a micro-nano fiber structure. CN 107923071 a discloses a vascular graft comprising a biodegradable scaffold and discloses a PGS core/PCL sheath structure, whose bioactivity is to be improved, and whose modulus is higher and whose compliance is to be improved. CN 109876192A discloses a bone repair membrane and a preparation method thereof, the bone repair membrane comprises an active layer, a barrier layer and a fixed layer, the materials of the active layer and the fixed layer are selected from one or more natural degradable materials, one or more synthetic degradable materials or the combination of two degradable materials; the natural degradable polymer is at least one of collagen, chitosan, gelatin, silk fibroin and hyaluronic acid; the synthetic degradable polymer is at least one of PLA, PLLA, PGA, PLGA, PGS and PHB. The above-mentioned bone repair membrane has a complicated structure, and the influence of the natural degradable polymer on the spinnability of PGA is not clear. CN 109295545A discloses a rigidity-controllable micro-nano oriented fiber, which is provided with a shell layer and a core layer, wherein the shell layer is prepared by using an elastic polymer and polyethylene oxide (PEO) blended solution, and the core layer is prepared by using a rigid polymer and polyethylene oxide. Whether the micro-nano oriented fiber has good film forming property or not and how to form the film is unknown, and when the micro-nano oriented fiber is used in vivo, PEO can be dissolved in water due to the fact that the environment in vivo is a water environment, and the dissolution or degradation of the fiber is caused.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a PGS/SF electrospun artificial blood vessel and a preparation method thereof, wherein the spinnability and fibroblasticity of PGS are improved by utilizing SF, the PGS/SF electrospun artificial blood vessel is successfully obtained, the caliber of the electrospun artificial blood vessel is less than 6mm, the electrospun artificial blood vessel has excellent elasticity of PGS and excellent biological and mechanical properties of SF, the electrospun artificial blood vessel still has a good micro-nano fiber structure after being cured, has adjustable inner diameter size and mechanical properties, and has the advantages of adjustable elasticity, biocompatibility, biodegradation and the like, and shows great application potential in the field of tissue engineering artificial blood vessels.

The invention aims to provide a preparation method of a PGS/SF electrospun artificial blood vessel, which comprises the following steps:

dissolving PGS prepolymer and SF in an organic solvent to obtain spinning solution, carrying out electrostatic spinning on the spinning solution, receiving the micro-nano-scale fibers by adopting a cylindrical receiving device rotating around the axis of the receiving device, winding the micro-nano-scale fibers outside the receiving device to form a tubular PGS/SF electrospun membrane, and drying and curing the tubular PGS/SF electrospun membrane after the spinning is completed to obtain a PGS/SF electrospun artificial blood vessel; the mass ratio of the PGS prepolymer to the SF is 0.1-100: 0.1-100;

the PGS prepolymer is polymer of sebacic acid and glycerol, the polymerization degree of the PGS prepolymer is 1-100, and the number average molecular weight M_n300-6000, weight average molecular weight M_wIs 1000-30000.

Further, the preparation method of the PGS prepolymer comprises the following steps:

heating sebacic acid and glycerol with equal molar ratio at the temperature of 120-140 ℃ in a protective atmosphere until the monomers are completely melted and uniformly mixed, and then reacting at the temperature of 120-140 ℃ for 24-48h to obtain the PGS prepolymer.

Further, the preparation method of SF comprises the following steps:

boiling silk in alkaline solution to remove sericin to obtain SF fiber, treating SF fiber in lithium bromide for 4-6h, dialyzing the obtained solution for 3 days to remove lithium bromide, centrifuging and drying to obtain SF.

Further, the molecular weight cut-off during dialysis was 3500 Da. By adopting the molecular weight cutoff, the molecular weight of SF in the PGS/SF electrospun artificial blood vessel prepared by the invention is more than 3500 Da.

Further, the mass ratio of the sum of the PGS prepolymer and SF in the spinning solution (i.e., the concentration of the spinning solution) is 6% to 16%. Preferably, the mass ratio of the sum of the mass of the PGS prepolymer and SF to the mass of the spinning dope is 8% to 10%, more preferably 9%. If the concentration of the spinning solution is too low, fibers cannot be formed, and if the concentration of the spinning solution is too high, the viscosity of the solution is too high, so that the polymer solution is gathered at a needle head, and the spinning is difficult.

Further, the organic solvent is one or a combination of more than two of hexafluoroisopropanol, dichloromethane, trichloromethane, tetrahydrofuran, methanol, formic acid, acetic acid, dimethyl sulfoxide, N-dimethylformamide and acetone. Preferably hexafluoroisopropanol. As SF is slowly dissolved in hexafluoroisopropanol, the dissolving time of PGS and SF in hexafluoroisopropanol at normal temperature needs 2-3 days, and the dissolving speed can be accelerated by heating and stirring.

Further, the voltage of electrostatic spinning is 12-15kV, the receiving distance is 12-15cm, the flow rate of spinning solution is 1-2mL/h, and the electrostatic spinning is carried out under the condition that the humidity is 30-50%.

Preferably, during electrostatic spinning, according to the quality of the spinning solution required by each electrostatic spinning, an injector with a proper size is mounted on the injection pump and used for injecting the electrospinning solution, the injector connected with the 18G needle is connected with the 21G needle by an 18S Teflon sleeve, the conductive wire at one end of the high-voltage electrostatic generator is connected with the 21G needle to form a high-voltage electric field, and the receiving device is a square plate covered with aluminum foil.

Further, in order to remove the electrospun membrane from the receiving device, the surface of the cylindrical receiving device was coated with a PEO membrane before electrospinning; after spinning is completed, the receiving device with the tubular product is soaked in water to remove the PEO film inside the tubular PGS/SF electrospun membrane and to detach the tubular PGS/SF electrospun membrane from the receiving device.

Further, a PEO aqueous solution with the mass ratio of 7.5% can be coated on the surface of the receiving device in advance through electrostatic spinning, and the PEO spinning time is 3-5 min.

Furthermore, the outer diameter of the cylindrical receiving device is 1-6mm, the length is 5-20cm, and the rotating speed is 20-1000 r/min.

And further, after spinning is completed, placing the spun electrospun membrane in a fume hood overnight, taking out the electrospun membrane, and placing the electrospun membrane in a vacuum drying oven to be cured at a certain temperature for a certain time to obtain PGS/SF membranes cured under different conditions.

Furthermore, the curing temperature is 120-140 ℃, and the curing time is below 24 h. Different curing temperatures can affect the curing rate and the curing degree of the PGS micro-nano fibers, so that the crystal structures in the micro-nano fibers are different, and the comprehensive performance of the prepared PGS/SF electrospun artificial blood vessel is finally affected.

Further, the mass ratio of the PGS prepolymer to SF is 3-7: 3-7. Preferably, the mass ratio of PGS prepolymer to SF is 7:3, 1:1, 3: 7. When the content of the PGS prepolymer is too high, the solution viscosity is too high, and it is difficult to perform electrospinning, or even if electrospinning can be performed only with difficulty, the electrospun fibers are severely bonded after they are received by the receiving device, and it is difficult to obtain an artificial blood vessel having a micro-nano-sized fiber. When the content of the PGS prepolymer is too low, the elasticity and mechanical properties of the artificial blood vessel are poor, which affects its practical application.

Further, the diameter of the micro-nano fibers is 1.5 μm or less. The inner diameter of the PGS/SF electrospun artificial blood vessel is 1-6 mm.

Further, the above preparation method further comprises a step of grafting heparin.

The second purpose of the invention is to provide the PGS/SF electrospun artificial blood vessel prepared by the preparation method, which comprises a tubular PGS/SF electrospun membrane, wherein the tubular PGS/SF electrospun membrane comprises a plurality of micro-nano fibers, and the micro-nano fibers comprise a PGS prepolymer and SF.

In the PGS/SF electrospun artificial blood vessel, the mass ratio of the PGS prepolymer to SF is 0.1-100:0.1-100, the PGS prepolymer is a polymer of sebacic acid and glycerol, the polymerization degree of the PGS prepolymer is 1-100, and the number average molecular weight M is_n300-6000, weight average molecular weight M_wIs 1000-30000. The diameter of the micro-nano fibers is less than 1.5 μm.

Further, heparin is grafted on the surface of the PGS/SF electrospun artificial blood vessel.

By the scheme, the invention at least has the following advantages:

(1) since PGS prepolymer alone cannot be sprayed by electrospinning to form fibers, PGS/SF electrospun artificial blood vessels, which are small-caliber artificial blood vessels, are successfully prepared by incorporating an SF film having good fiberizability. The artificial blood vessel preparation technology is simple and convenient to operate and can be used for large-scale production.

(2) The PGS/SF electrospun artificial blood vessel has a good micro-nano fiber structure, can simulate a three-dimensional network structure of extracellular matrix to a greater extent, and promotes the adhesion and proliferation of cells. And SF is natural polymer fibrin, and can contribute to improving the bioactivity of the electrospun membrane.

(3) The PGS/SF electrospun artificial blood vessel has better mechanical property, and the PGS/SF electrospun artificial blood vessel has adjustable mechanical property by changing the curing time.

(4) The PGS/SF electrospun artificial blood vessel has the compliance matched with a natural blood vessel, and meanwhile, the degradation rate is controllable, so that the PGS/SF electrospun artificial blood vessel is expected to realize the successful transplantation of the small-caliber artificial blood vessel.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to clearly understand the technical solutions of the present invention and to implement the technical solutions according to the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the detailed drawings.

Drawings

FIG. 1 is SEM characterization results of PGS/SF electrospun membranes prepared at different spinning solution concentrations;

FIG. 2 shows the macro morphology of PGS/SF electrospun tubing prepared by different mass ratios of PGS and SF after being cured for 24 hours at 130 ℃;

FIG. 3 is a scanning electron microscope image of PGS/SF electrospun tubing prepared by different mass ratios of PGS and SF, cured at 130 ℃ for 24 h;

FIG. 4 is FTIR-ATR spectra before and after curing of PGS/SF electrospun tubing at different mass ratios;

FIG. 5 is an X-ray diffraction curve of PGS/SF electrospun tubing of different mass ratios before and after curing;

FIG. 6 is a stress-strain curve for PGS/SF electrospun tubing of different mass ratios;

FIG. 7 shows the results of the suture retention strength test of PGS/SF tubular materials with different mass ratios;

FIG. 8 shows the results of compliance testing of PGS/SF tubular materials of different mass ratios;

FIG. 9 shows the results of testing the cyclic tensile properties of PGS/SF tubular materials with different mass ratios;

FIG. 10 shows the results of tensile property tests of PGS/SF tubular materials with different mass ratios under different curing conditions;

FIG. 11 is FTIR-ATR spectra of PGS/SF electrospun material and heparin-grafted PGS/SF electrospun material;

FIG. 12 shows the BCI values for grafted heparin and grafted heparin for different ratios of PGS/SF composite scaffolds and PLCL scaffolds;

FIG. 13 is a graph of the change in mass over a 5 week period of degradation for different samples;

FIG. 14 is SEM characterization results after 5 weeks of degradation of different samples;

FIG. 15 is a laser confocal image of human umbilical vein endothelial cells cultured on different electrospun membranes for 1-7 days;

FIG. 16 is a graph of MTT proliferation of human umbilical vein endothelial cells cultured on different electrospun membranes for 1-7 days.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1

(1) Synthesis of PGS prepolymer: adding 0.1mol of sebacic acid and 0.1mol of glycerol into a 250mL three-necked flask, placing the flask in a magnetic stirrer, stirring and heating for reaction, introducing nitrogen at normal temperature, heating in an oil bath, stirring and heating at 125 ℃ for reaction for 4 hours, and completely melting and uniformly mixing the monomers. The reaction was continued for 48h while maintaining the temperature under nitrogen atmosphere to obtain PGS prepolymer. Cooling to room temperature to obtain white waxy solid, and storing at 4-8 deg.C.

The synthesis route of PGS is shown below, where n is 6-50:

(2) preparation of SF film: weighing 21.2g of anhydrous sodium carbonate, dissolving in 10L of heated and boiled deionized water, degumming 25g of raw silkworm silk for 30min at 100 ℃ under boiling condition, taking out after degumming, washing with deionized water for five times, and removing residual sericin. And (5) drying the split yarn in an oven at 60 ℃ to obtain the pure SF fiber. The SF fibers were removed from the oven and were dried at a bath ratio of 2.7: 10 was dissolved in 9.3M lithium bromide solution and the beaker was sealed and dissolved for 4-5h in an oven at 60 ℃. Cooling to room temperature, placing into dialysis bag (molecular weight cut-off 3500Da), sealing, and dialyzing with deionized water for 3 days to remove lithium bromide. And centrifuging the dialyzed SF solution for 20min by a high-speed centrifuge at the rotating speed of 9000r/min to obtain the SF solution. The obtained SF solution is formed into a film at normal temperature, so that the solvent in the SF solution is removed.

(3) PGS and dried SF with the mass ratio of 1:1 are respectively weighed by a precision balance, the PGS and the dried SF are dissolved in Hexafluoroisopropanol (HFIP), the mixture is magnetically stirred for 1-2 days at the temperature of 35 ℃ until no particles are visible in the spinning solution, and a plurality of groups of experiments are carried out in parallel, so that the mass of solute (the sum of the mass of PGS and the mass of dried SF) respectively accounts for 16%, 14%, 12%, 10%, 9%, 8%, 7% and 6% of the total weight of the spinning solution. According to the quality of spinning solution required by each electrostatic spinning, a syringe with a proper size is installed on an injection pump and used for sampling the electrospinning solution, the syringe connected with an 18G (phi 0.84 multiplied by 1.27mm) needle is connected with a 21G (phi 0.51 multiplied by 0.82mm) needle by using an 18S (phi 1.07 (inner diameter) × 1.87 (outer diameter) mm, S-shaped) teflon sleeve, a conductive wire at one end of a high-voltage electrostatic generator is connected with the 21G (phi 0.51 multiplied by 0.82mm) needle to form a high-voltage electric field, and a receiving device is a square plate covered with aluminum foil. The voltage used in this experiment was 12.5-15kV, the receiving distance was 12cm, and the flow rate was 2 mL/h.

(4) And (3) placing the spun electrostatic spinning membrane in a fume hood overnight, taking out the electrostatic spinning membrane, and placing the electrostatic spinning membrane in a vacuum drying oven for drying treatment at 60 ℃ to obtain different PGS/SF electrospun membranes.

FIG. 1 is SEM characterization results of PGS/SF electrospun membranes prepared at different spinning solution concentrations. The concentration of the spinning solution has great influence on the spinning state and the fiber appearance during electrostatic spinning, when the concentrations of the spinning solutions are 16% (figure 1a), 14% (figure 1b) and 12% (figure 1c), the solution viscosity is increased along with the increase of the concentrations of the spinning solutions, the spinning solutions are easily gathered on a needle head, the spinning is difficult, large fibers of an electrospinning material are easily in a mixed adhesion state, the thickness of the electrospinning fiber is uneven, the fiber diameter is generally thick, and the fiber diameter does not have statistical significance. When the concentration of the spinning solution was 10%, the needle was easily clogged with the spinning solution, but large-area blocking of the fibers did not occur, the fiber thickness was uniform, and the average fiber diameter was 0.838. mu.m (FIG. 1 d). When the concentration of the spinning solution is 9%, the spinning process is smooth, the needle head is not blocked, the fiber is well formed and has uniform thickness, and the average diameter of the fiber is 0.781 μm (figure 1 e). At a dope concentration of 8%, occasionally, droplets were ejected, affecting the fiber morphology, and the average fiber diameter was about 0.776 μm (FIG. 1 f). When the concentration of the spinning dope was further decreased to 7%, it was found that the spinning dope was ejected substantially in the form of droplets, and the fiber formation was poor (FIG. 1 g).

In conclusion, when the spinning solution concentration is 9%, the fibrilization of PGS in the electrospun membrane is better.

Example 2

An artificial blood vessel was prepared by following the method of steps (1) to (2) of example 1 by selecting a spinning solution with a concentration of 9% and changing the mass ratio of PGS to SF to 70/30, 50/50 and 30/70, and in step (3), the receiving device was changed to a stainless steel roller with a diameter of 4mm, and in order to facilitate the removal of the sample from the stainless steel, a PEO aqueous solution with a mass ratio of 7.5% was previously spun on the stainless steel roller for a PEO spinning time of 3 to 5 min. The electrospinning parameters were set as follows: the voltage is 12.5-15kV, the receiving distance is 12cm, the flow rate is 2mL/h, the spinning time is 1-1.5h, and the rotating speed is 500 r/min. The micro-nano-scale fibers generated in the electrostatic spinning process are wound outside the receiving device to form a tubular PGS/SF electrospun membrane, the receiving device with the tubular PGS/SF electrospun membrane is immersed in water after the spinning is completed to remove a PEO layer, and the PGS/SF electrospun pipe can be smoothly demoulded.

Since PGS has adjustable mechanical properties, it can be adjusted by curing temperature and curing time. The curing temperature is set at 130 ℃ and the curing time is 24 h. And (3) placing the spun sample in a fume hood overnight to remove redundant hexafluoroisopropanol, taking out the sample, and placing the sample in a vacuum drying oven for curing to obtain the cured PGS/SF electrospun pipe.

FIG. 2 shows the macro-morphology of PGS/SF electrospun tubing prepared from PGS and SF with different mass ratios after being cured for 24h at 130 ℃. In fig. 2, the small-bore pipe was yellowish in color after curing, and the color was darker as the PGS ratio was increased, which may be due to the characteristics of PGS. The PGS/SF electrospun pipe before and after curing has small caliber change and good shape retention, and the inner diameter of the PGS/SF electrospun pipe after curing is about 3.883 mm.

FIG. 3 is a scanning electron microscope image of PGS/SF electrospun tubing prepared by different mass ratios of PGS and SF, which is cured at 130 ℃ for 24h, and the three types of PGS/SF electrospun artificial blood vessels still have good micro-nano fiber structures after being cured. However, the PGS is easy to melt during the curing process to generate a flow phenomenon, so that the surface of the PGS/SF (70/30) pipe (FIGS. 3a1 and a2) has more fiber adhesion, but the fiber fineness is finer, and the average diameter of the fiber is 0.558 mu m. The fiber diameter coarsened as the SF ratio increased, with PGS/SF (50/50) tubing having a fiber mean diameter of 0.763 μm (FIGS. 3b1, b2) and PGS/SF (30/70) tubing having a fiber mean diameter of 0.962 μm (FIGS. 3c1, c2), since the addition of SF increased the viscosity of the dope, preventing the jet from drawing by coulomb force, and therefore coarsening of the fiber diameter. The wide fiber diameter distribution is because secondary spinning jets are jetted from the primary spinning jets during spinning, and depending on the spinning conditions, a small portion of fibers having a smaller diameter or a larger diameter are produced.

The PGS/SF electrospun pipes prepared in the mass proportions have certain degrees of pore structures, the porosity of the PGS/SF (30/70) pipe is 53.41 +/-0.9% larger, the porosity of the PGS/SF (50/50) is 40.83 +/-2.8%, and the porosity of the PGS/SF (70/30) is 21.91 +/-1.1% lower. The increase in the proportion of PGS caused a decrease in the porosity of the fibers at the surface of the sample due to the melt flow of PGS during solidification, while the addition of SF increased the porosity of the material.

FTIR-ATR spectra before and after curing of PGS/SF electrospun tubing of different mass ratios are shown in FIG. 4. In the figure, a is a sample of PGS/SF-0/100, and b is a tubular sample of PGS/SF-70/30C is a tubular material with PGS/SF of 50/50, d is a tubular material with PGS/SF of 30/70, and a '-e' is a tubular material obtained by curing a-e. After curing, the characteristic absorption peaks of PGS and SF can still be found. PGS/SF films of each mass ratio were found at 1744cm^-1、1165cm^-1、1650cm^-1、1622cm^-1、1515cm^-1、1223cm^-1Has obvious characteristic absorption peaks. 1744cm^-1C ═ O stretching vibration peak at 1165cm belonging to ester carbonyl group^-1Is the C-O-C asymmetric stretching vibration peak of ester, 3458cm^-1The broad peak at (a) is the stretching vibration peak of the associated hydroxyl group. After curing, the ester group C ═ O bonds were condensed (1744 cm)^-1) The tensile peak intensity increased and originated from the-OH bond (3458 cm)^-1) The peak of (a) was reduced in intensity, indicating an increase in cross-link density of the PGS, thus indicating that an increase in temperature contributes to curing of the PGS. At 1622cm^-1、1515cm^-1、1223cm^-1The absorption peaks are respectively the characteristic absorption bands of amide I, amide II and amide III of the silk fibroin, and the curing condition has no influence on the secondary structure of the silk fibroin.

The X-ray diffraction curves of PGS/SF electrospun pipes with different mass ratios before and after solidification are shown in FIG. 5. In the figure, a is a sample of PGS/SF-0/100, b is a tubular material of PGS/SF-30/70, c is a tubular material of PGS/SF-50/50, d is a tubular material of PGS/SF-70/30, and a '-e' are cured tubular materials of a-e, respectively. From the figure, it can be found that the pure SF sample has a stronger diffraction peak at 19.9 degrees and 24.3 degrees respectively, a silk II structure at 19.9 degrees and a silk I structure 60 at 24.3 degrees. With a smaller SF ratio, the silk I structure in the PGS/SF tubular material disappears, and the silk II structure exists mainly. After curing, the silk II structure in the PGS/SF tubular material still exists, and the diffraction peak of SF is weakened along with the increase of PGS. PGS belongs to the class of semi-crystalline polymers, i.e. polymers that cannot be completely or partially crystallized, in which the polymers are present in a mixture. XRD demonstrated the presence of secondary fibroin structures, but did not well demonstrate the presence of PGS after curing, indicating that PGS also exhibits an amorphous structure after curing.

When the contact angle is between 0 and 90 deg., the material is hydrophilic. The contact angles of the PGS/SF electrospun pipes in the mass ratios are tested, and the results show that the surface contact angles of PGS/SF (70/30), PGS/SF (50/50), PGS/SF (30/70) and PGS/SF (0/100) are respectively 48 degrees, 0 degree and 0 degree, and the excellent hydrophilicity is shown. PGS is not hydrophilic enough, and the addition of SF can improve the hydrophilicity well.

The tensile properties of the samples prepared in the above examples were tested, and the results are shown in table 1, and it is known that the human internal mammary artery rupture strength is 3.1MPa, the elongation at break is 134%, and the young's modulus is 0.26-8MPa, and when the PGS ratio reaches 50% or more, the PGS/SF tubular material exhibits better mechanical properties than the human internal mammary artery. With the increase of the PGS proportion, the breaking strength and the breaking elongation are both improved, and the Young modulus is reduced. The stress-strain curves of the above examples are shown in fig. 6.

Table 1: different PGS/SF tubular material thickness, breaking strength, breaking elongation and Young modulus

The artificial blood vessel is clinically used and needs to have certain suture retention strength, which is the force required by pulling out the surgical suture from the artificial blood vessel, so as to ensure that the artificial blood vessel and the natural blood vessel are sutured together without causing rupture of the blood vessel. The suture retention strength of the PGS/SF tubular material at each mass ratio is shown in FIG. 7. The suture retention strength of PGS/SF (70/30) and PGS/SF (50/50) tubular materials is obviously superior to that of PGS/SF (30/70), which is consistent with the previous radial tensile property result, and the PGS plays a great role in improving the mechanical property of the stent after being cured along with the increase of the proportion of the PGS. The suture retention strength of PGS/SF (70/30) reaches 2N on average, and reaches the requirement of suture retention strength required by artificial blood vessel implantation, the value of the suture retention strength of the PGS/SF is close to that of human saphenous vein suture retention strength of 1.72-2.62N, and is far greater than that of human internal mammary artery of 1.35-1.4N.

The artificial blood vessel and the host blood vessel are not matched in compliance, the hemodynamic state of blood is changed, so that the artificial blood vessel transplantation fails due to the problems of thrombus, intimal hyperplasia and the like. The artificial blood vessel compliance at each mass ratio is shown in fig. 8. The PGS content is different, the compliance difference is large, and the compliance value of the PGS/SF (70/30) tubular material is as high as 3.58%/100 mmHg, which is far beyond the compliance value of the commercial artificial blood vessel. The compliance values decreased with decreasing PGS ratios, with PGS/SF (50/50, 30/70) tubular stents having compliance values of 1.45%/100 mmHg, 1.34%/100 mmHg, approaching those of the human saphenous vein by 0.7-5.0%/100 mmHg.

The elastic characteristics of the pipes prepared according to the invention were investigated by means of radial cyclic tensile tests. The cyclic stretching performance of the PGS/SF tubular samples in each mass ratio is shown in fig. 9, wherein a is a PGS/SF (70/30) electrospun tubular material, B is a PGS/SF (50/50) electrospun tubular material, C is a PGS/SF (30/70) electrospun tubular material, D is a PLCL electrospun tubular material, and E, F is cyclic stretching for one time and 50 times respectively for the four samples. PGS/SF tubular samples of different mass ratios all show excellent cycling behavior with low energy dissipation per cycle. After 50 stretching cycles, a small amount of stress relaxation phenomenon occurs in the PGS/SF electrospun pipe, the stress of the PGS/SF (70/30) electrospun pipe is reduced to 0.1MPa, the stress of the PGS/SF (50/50) electrospun pipe is reduced to about 0.1MPa, and the stress of the PGS/SF electrospun pipe is reduced to about 0.05 MPa. The stress of the PLCL electrospun pipe is reduced by about 0.3MPa, larger stress relaxation is generated, and the area formed by the hysteresis curve of the PLCL electrospun pipe is much larger than that of the PGS/SF electrospun pipe, which shows that the PGS/SF electrospun pipe has better cyclic tensile property and elasticity than that of the PLCL electrospun pipe.

Example 3

Pipes were prepared according to the method of example 2, using a mass ratio of PGS and SF of 70/30, 50/50, 30/70, except that curing was carried out at 120, 125, 130, 135, 140 ℃ for 24 h. FIG. 10 shows the tensile properties of PGS/SF tubular materials at various mass ratios under different curing conditions. When the temperature is lower than 130 ℃, the mechanical property of the PGS/SF tubular material is poor, and the PGS curing rate is low; when the curing temperature is raised to 130 ℃, the mechanical property of the PGS/SF tubular material is obviously improved, and the breaking strength and the breaking elongation are improved along with the further improvement of the curing temperature.

Example 4

When the small-caliber artificial blood vessel has insufficient blood compatibility, and because the blood flow volume passing through the inner cavity of the small-caliber artificial blood vessel is small and the flow rate is slow, thrombus is easily formed on the cavity surface, and thus the blood vessel occlusion occurs to cause the transplantation failure. Heparin is a commonly selected anticoagulant, and a large amount of sulfate radicals exist in molecules of the heparin, and the sulfate radicals have a large amount of negative charges and are related to the anticoagulant effect of the heparin. The invention selects heparin to carry out surface anticoagulation treatment on the small-caliber artificial blood vessel material so as to improve the blood compatibility of the small-caliber artificial blood vessel material.

200mg Heparin (HP) was added to 100mL of 0.05N MES (pH 5.5) solution containing 0.1N HCl, 200mg EDC and 120mg NHS and activated at 4 ℃ for 4 h. Immersing the stent in an activated heparin (hp) solution for grafting reaction, reacting amino in SF with NHS on the activated heparin, carrying out ultrasonic washing for 3 times by deionized water after overnight at 37 ℃ to obtain the heparinized small-caliber artificial blood vessel stent.

The principle of the heparin activation reaction is as follows:

the heparin grafting reaction principle is as follows, wherein H₂P or PH₂Represents grafted heparin:

FIG. 11 is FTIR-ATR spectra of PGS/SF electrospun material and PGS/SF electrospun material grafted with heparin. PGS/SF electrospun material grafted with heparin at 820cm^-1、942cm^-1、1038cm^-1Has a characteristic absorption peak at 820cm^-1Is the C-O-S stretching vibration peak of sulfate group on the hexosamine, 942cm^-1A heparin characteristic absorption peak; 1038cm^-1Is SO of sulfonic acid₂Group characteristic peak position. Heparin was demonstrated to have been grafted onto the samples.

Tables 2-3 show that the hemolysis rates of the PGS/SF electrospun material and the PLCL electrospun material with different mass ratios before and after heparin grafting are both below 5%, the hemolysis rate of the material after heparin grafting is reduced, and the blood compatibility of heparin to the material is improved.

Table 2: results of hemolysis test of groups of samples without grafted heparin

Table 3: hemolysis test results for each set of samples grafted with heparin

FIG. 12 shows the BCI values for grafted heparin and grafted heparin for different ratios of PGS/SF composite scaffolds and PLCL scaffolds. As can be seen, the BCI values of PGS/SF (70/30), PGS/SF (50/50), PGS/SF (30/70), PGS/SF (0/100) and PLCL scaffolds not grafted with heparin were 74.65%, 75%, 78.44%, 76.72%, 70.9%, respectively, and the BCI values of PGS/SF (70/30), PGS/SF (50/50), PGS/SF (30/70), PGS/SF (0/100) and PLCL scaffolds grafted with heparin were 91.97%, 89.32%, 91.3%, 83.25%, 79.79%, respectively. The BCI value of the material grafted with heparin is obviously increased, the anticoagulation effect is positively correlated with the BCI value, and the anticoagulation effect is increased.

Example 5

The electrospun material prepared in example 2 was subjected to a degradation experiment, the PLCL electrospun material was a control sample, the sample was cut to a length of about 1mm and placed in a 96-well plate, about 300 μ L of PBS buffer (pH 7.4) was added to each well, and the 96-well plate was placed in a constant temperature shaking water bath at 37 ℃ to simulate an in vivo dynamic environment and observed for one month. Fresh PBS buffer was replaced weekly.

The change in mass over the 5 week period of degradation for each set of samples is shown in figure 13. After 5 weeks of degradation time, the weight loss rate of the pure SF membrane is only about 5%, the pure SF membrane is hardly degraded before 4 weeks, and the SF membrane has relatively stable degradation property in vitro. The PGS/SF electrospun membranes with different mass ratios have different degradation behaviors, and the increase of the PGS ratio can increase the degradation rate of the sample and obviously increase the weight loss rate. The PGS/SF (70/30) electrospun material reached a weight loss rate of 19% at a degradation time of five weeks, which was also consistent with its pH value being substantially reduced at week 5. The PGS/SF (50/50) electrospun material had a faster degradation rate at week 4, with a weight loss rate within 5 weeks that was generally consistent with the PGS/SF (70/30) electrospun material. The PGS/SF (30/70) electrospinning material has stable degradation behavior within 4 weeks, and the weight loss rate within 5 weeks is about 10%. The addition of SF slows down the degradation rate of PGS in the PGS/SF electrospun membrane, and the compounding of the synthetic material and the natural material is beneficial to maintaining the quality of the sample and slowing down the excessive degradation of the sample. The weight loss rate of the PLCL scaffold in 5 weeks is about 4%, and the degradation rate is relatively slow.

The morphology of each group of samples after 5 weeks of degradation is shown in fig. 14, fig. 14a-e are SEM test results of pure SF electrospun membrane, pure PLCL electrospun membrane, PGS/SF (70/30) electrospun membrane, PGS/SF (50/50) electrospun membrane, and PGS/SF (30/70) electrospun membrane in this order. As can be seen from the figure, after 5 weeks of degradation, the diameters and the shapes of the electrospun materials of SF and PLCL are not obviously changed, and the phenomenon of fiber adhesion appears on the surface of the electrospun material of PGS/SF, which is probably due to slight hydrolysis of the fibers, and the adhesion phenomenon is obvious along with the increase of the proportion of PGS, but the phenomenon of fiber breakage does not appear yet.

Example 6

The electrospun material prepared in example 2 was subjected to in vitro cell compatibility evaluation. The results of culturing HUVECs cells on PGS/SF electrospun membranes and PLCL electrospun membranes at different mass ratios are shown in FIG. 15.

The next day of cell inoculation, the cells are more evenly dispersed on the surface of each group of materials, and the cells grow along the fibers in a spindle shape. By the fourth day, the proliferation of cells on each group of scaffolds is obviously increased, the cells can still be seen to grow along the fiber direction, and a small amount of sheet-shaped adhesion phenomenon begins to appear on the cells on the surface of the PGS/SF electrospun material, which may be caused by the sheet-shaped area formed by melting of the PGS on the surface of the solidified PGS/SF electrospun material or by the cell proliferation which is too fast, so that the cells form sheet-shaped adhesion. After 7 days of cell seeding, the surface of each group of materials begins to show large areas of cell adhesion, endothelialization begins to appear on the surface of the materials, but the cells on the surface of the pure SF material still keep clear veins and are well-defined, and the cells can grow orderly along the sequence of fibers on the surface of the material, which is probably caused by the higher porosity of the pure SF.

The growth of HUVEC cells on the surface of the material was quantified by MTT. As shown in fig. 16, the cells on the surfaces of PGS/SF electrospun material and PLCL electrospun material with different mass ratios showed a trend of increasing significantly with the increase of the number of days of culture, and the cell growth on each group of samples was not significantly superior, but slightly lower than that of the cells on the blank culture plate. Proved that all groups of samples have good cell compatibility and can well support the spreading and proliferation of HUVEC cells.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of PGS/SF electrospun artificial blood vessel is characterized by comprising the following steps:

dissolving PGS prepolymer and SF in an organic solvent to obtain spinning solution, carrying out electrostatic spinning on the spinning solution, receiving micro-nano-scale fibers by adopting a cylindrical receiving device rotating around the axis of the receiving device, winding the micro-nano-scale fibers outside the receiving device to form a tubular PGS/SF electrospun membrane, and drying and curing the tubular PGS/SF electrospun membrane after the spinning is completed to obtain the PGS/SF electrospun artificial blood vessel; the mass ratio of the PGS prepolymer to SF is 0.1-100: 0.1-100;

the PGS prepolymer is a polymer of sebacic acid and glycerol, and the polymerization degree of the PGS prepolymer is 1-100.

2. The method of claim 1, wherein the PGS prepolymer is prepared by the steps of:

and melting sebacic acid and glycerol in equal molar ratio at the temperature of 120-140 ℃ in a protective atmosphere, reacting for 24-48h to obtain the PGS prepolymer.

3. The method of claim 1, wherein the SF is prepared by the steps of:

boiling silk in an alkaline solution to remove sericin to obtain SF fiber, treating the SF fiber in lithium bromide for 4-6h, dialyzing the obtained solution to remove lithium bromide, centrifuging and drying to obtain SF.

4. The method according to claim 3, wherein the cut-off molecular weight is 3500Da during dialysis.

5. The production method according to claim 1, wherein the mass ratio of the sum of the mass of the PGS prepolymer and the mass of SF in the spinning solution is 6% to 16%.

6. The method according to claim 1, wherein the electrospinning voltage is 12 to 15kV, the take-up distance is 12 to 15cm, the flow rate of the spinning solution is 1 to 2mL/h, and the electrospinning is carried out under a humidity of 30 to 50%.

7. The method as claimed in claim 1, wherein the curing temperature is 120-140 ℃.

8. The method of claim 1, wherein prior to electrospinning, the surface of the cylindrical receiving device is coated with a PEO film; after spinning is completed, the receiving device with the tubular product is soaked in water to remove the PEO film inside the tubular PGS/SF electrospun membrane and to detach the tubular PGS/SF electrospun membrane from the receiving device.

9. The production method according to claim 1, wherein the diameter of the micro-nano-sized fiber is 1.5 μm or less; the inner diameter of the PGS/SF electrospun artificial blood vessel is 1-6 mm.

10. A PGS/SF electrospun artificial blood vessel prepared by the preparation method according to any one of claims 1 to 9, characterized in that: the PGS/SF electrospun membrane is tubular, the tubular PGS/SF electrospun membrane comprises a plurality of micro-nano fibers, and the micro-nano fibers comprise PGS prepolymer and SF.