CN112402690A - Woven reinforced degradable polyurethane elastomer artificial blood vessel and preparation method thereof - Google Patents
Woven reinforced degradable polyurethane elastomer artificial blood vessel and preparation method thereof Download PDFInfo
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- 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
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- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
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- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
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- D01D5/0069—Electro-spinning characterised by the electro-spinning apparatus characterised by the spinning section, e.g. capillary tube, protrusion or pin
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- D04C1/00—Braid or lace, e.g. pillow-lace; Processes for the manufacture thereof
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- D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
- D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
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- D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
- D06M15/19—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with synthetic macromolecular compounds
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- D06M2101/16—Synthetic fibres, other than mineral fibres
- D06M2101/30—Synthetic polymers consisting of macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- D06M2101/38—Polyurethanes
Abstract
The invention provides a woven reinforced degradable polyurethane elastomer artificial blood vessel and a preparation method thereof. The inner layer of the artificial blood vessel adopts activated H2N-PEG-COOH, heparin and (Mpa) -GGGGGREDV polypeptide pair C-PEEEUU-NH2Processing the electrospun nanofiber tubular scaffold to obtain the C-PEEEUU-PEG-Hep/REDV tubular scaffold, wherein the middle layer is a nanofiber yarn woven enhancement layer woven by dissolving and mixing one or more master batches of PLGA, PDO and PGA and silk fibroin according to a mass ratio, and the outer layer is a macroporous layer prepared by thermally induced phase separation. The woven enhanced degradable polyurethane elastomer artificial blood vessel provided by the invention can be used for tissue regeneration and construction of cardiovascular and cerebrovascular diseases, the preparation method is simple and efficient, the price is low, and the prepared artificial blood vessel has resilience, compliance, bursting strength and biocompatibility matched with autologous vascular tissues. Has no hypersensitivity to human body during transplantation operation, has affinity with blood and peripheral tissues in human body, and has good application prospect in heart surgery, nephrology surgery and brain surgery repairing operation.
Description
Technical Field
The invention relates to the field of artificial blood vessels, in particular to a woven reinforced degradable polyurethane elastomer artificial blood vessel and a preparation method thereof.
Background
According to the world health report of the World Health Organization (WHO), the death number of cardiovascular and cerebrovascular diseases in the world accounts for 30.3 percent of the total death population. In China, the morbidity and the mortality of cardiovascular and cerebrovascular diseases are the first of various diseases, and the morbidity and the mortality of the cardiovascular and cerebrovascular diseases in China are still rising continuously. According to the statistics of the Ministry of health, the number of cardiovascular and cerebrovascular diseases in China is 2.9 hundred million, and the data can break through 3.2 million by 2020. The most effective method for treating cardiovascular and cerebrovascular diseases is surgical transplantation, however, autografting and allograft transplantation have many defects. With the introduction of tissue engineering and regenerative medicine concepts, large-caliber artificial blood vessels made of materials such as PET and PTFE have been successfully used as blood vessel substitutes, but clinical use of small-caliber artificial blood vessels has many problems, such as early acute thrombosis, severe anastomotic intimal hyperplasia, and mechanical matching between the artificial blood vessel and autologous tissue.
In the process of using the artificial blood vessel stent for autologous vascular tissue repair, the performance of the material and the structure of the artificial blood vessel are two important factors influencing whether the blood vessel is successfully reconstructed. In recent years, many basic researches and clinical trials have been conducted around the development of a novel artificial blood vessel material and a preparation process of a novel artificial blood vessel. Polyurethane and silk powder are used as basic Materials to develop a high-bionic small-caliber artificial blood vessel (Xu WL, Zhou F, Ouyang CX, et al. mechanical properties of small-diameter polyurethane vascular tissue, which has the same structure and similar function as human blood vessels) (Xu WL, Zhou F, Ouyang CX, et al. mechanical properties of small-diameter polyurethane vascular tissue, second-dimension polyurethane vascular tissue, J. Journal of biological Materials Research Part A2010, 92A (1): 1-8; Europe morning, Chengxui, Cao jin, etc.. the once-formed polyurethane artificial blood vessel and the preparation method thereof [ P ]. Chinese invention patent, patent number: 201410071581.8, application date: 2014.2.28 ZL.) are developed. Although the used silk powder has good biocompatibility with autologous vascular cell tissues, the silk powder material lacks the elasticity and flexibility of human blood vessels in terms of mechanical properties. On the other hand, the group used polyurethanes as non-degradable materials, although the tubular scaffolds were prepared with biomimetic pore structures, such a persistent pore structure would not perfectly match the morphology of the host cells at various stages, all in all, the host cells would change their morphology with the pore structure, which would be detrimental to tissue regeneration. Mi et al prepared a three-dimensional tubular scaffold with an inner layer of TPU electrospun nanofibers, a middle layer of a silk fibroin braid and an outer layer of a TPU thermo-induced separation layer, although the scaffold well simulates a natural vascular structure and has mechanical properties meeting clinical requirements, the scaffold has a large number of layers and a large wall thickness, cannot be well matched with autologous vascular tissues in the transplanting process, has the risk of rapid proliferation at an anastomotic site, and has no bioactivity and biodegradability (MiH. -Y., Jing X., Yu E., et al. applied to biological to branched-layered vascular tissue repair methods [ J ]. Industrial & Engineering chemical Research,2016,55, 882-; patent application No. 201510953566.0 of a three-layer scaffold prepared by Mi Yang, Mi, Miyao, et al, application date: 2015.12.17.).
In recent years, with the research on materials such as artificial blood vessels such as PET and ePTFE, it has been found that such materials have the disadvantage of being highly hydrophobic, are not conducive to endothelialization and thus are likely to induce thrombus formation; the flexibility and the elasticity of the artificial blood vessel stent can not meet the requirements of tissue engineering blood vessel stents, the artificial blood vessel stent can not be degraded after being implanted into a body and can not remold blood vessel tissues, the long-term patency rate after being implanted is not ideal although the artificial blood vessel stent can be unobstructed in a short period after being implanted, and the defects are more obvious in the application of small-caliber artificial blood vessels. The advantage of degradable polyurethane for small-bore artificial blood vessels is becoming more prominent, and it was reported in a document of the Wagner project group of Pittsburgh university that a degradable polyurethane is synthesized based on PCL diol and 1, 4-diisocyanate or lysine diisocyanate as monomers and putrescine as a chain extender, and the polyurethane is electrospun into nanofibers, followed by surface amination and RGDS short-peptide grafting, and in vitro cell experiment results show that the survival rate of PEUU cells grafted with RGDS is increased by 10% to 30%, while the cell adhesion number is significantly increased and is 2 times of the cell number on PEUU nanofibers (Guan J., Sacks M.S., Beckman E.J., et al. Synthesis, chromatography, and catalysis of electrochemical, bioble polyurethane, biological polyethylene-polyurethane, 61,493-503.). Because the nanofiber has a compact pore structure, the proliferation and migration of host cells are not facilitated. Wagner and Vorp make PEUU through thermal phase separation and electrostatic spinning technology to prepare a double-layer vascular stent with a macroporous structure as an inner layer and a compact nanofiber layer as an outer layer, the composite artificial blood vessel has good mechanical matching property, but the nanofiber layer on the outer layer blocks adventitia stem cells and capillaries in peripheral/peripheral tissues of the blood vessel, and the adventitia stem cells and the capillaries can permeate through the wall of the artificial blood vessel and infiltrate into the vascular stent to promote angiogenesis. It is also that such vascular prostheses do not facilitate the maintenance of blood flow homeostasis due to the roughness of the inner surface compared to the outer surface, since the turbulent flow of blood due to the wall roughness is also a cause of thrombus formation (Soletti L., Hong Y., Guan J., et al. A bilayered elastomeric scaffold for tissue engineering of small diameter vascular grafts [ J ]. Acta biomaterials, 2010,6, 110-. The research on the synthesis of degradable polyurethane using PCL diol as monomer and its application in small vessel tissue engineering has never been interrupted in the last two decades, however most of the research only has been on the level of modification of Materials and in vitro cell experiments, and even some in vivo transplantation research can not obtain good results due to acute coagulation or proliferation problems (Fang J., Zhang J.L., Du J., et al. organic functional polyurethane with sub-tissue modification with side and end-absorbing peptide expression for vascular recovery [ J ]. Applied Materials & Interfaces,2016,8,14442 and 14452.). So far, reports of preparing small-caliber artificial blood vessels by adopting a method of combining an electrostatic spinning technology, a weaving technology and a thermally induced phase separation technology do not appear. Therefore, the three technologies are combined with material modification synthesis and functional modification to prepare the multi-stage functional structure artificial blood vessel which has mature process, close fit of the inner layer and the outer layer, no layering phenomenon, good mechanical matching and excellent biocompatibility, and has great economic benefit.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a woven reinforced degradable polyurethane elastomer artificial blood vessel and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a braided reinforced degradable polyurethane elastomer artificial blood vessel, which comprises the following steps:
mixing and dissolving one or more master batches of PLGA (polylactic acid-glycolic acid copolymer), PDO (1, 3-propylene glycol) and PGA (polyglycolic acid) to obtain a composite spinning solution;
spinning the composite spinning solution by adopting a double-needle-head opposite-spraying electrostatic spinning system to obtain nanofiber yarns, and weaving the nanofiber yarns into tubular braided fabrics;
spinning PEO (polyethylene oxide) nano-fibers on the stainless steel of the die by an electrostatic spinning technology, and then spinning C-PEEEUU-NH2A nanofiber; loading PEO and C-PEEEUU-NH2Putting the stainless steel of the nano-fiber into ultrapure water for demolding; removing the removed C-PEEEUU-NH2Continuously stirring and cleaning the nanofiber tubular support in ultrapure water until impurities on the surface of the nanofiber tubular support are removed, and then carrying out vacuum freeze drying;
step four, adopting activated H2N-PEG-COOH, heparin and (Mpa) -GGGGGREDV polypeptide on the C-PEEEUU-NH2Grafting and modifying the nanofiber tubular scaffold to obtain a C-PEEEUU-PEG-Hep/REDV nanofiber tubular scaffold;
step five, sheathing the shaft core of a special mould with the C-PEEEUU-PEG-Hep/REDV nano fiber tubular bracket, and then drying;
step six, sleeving the tubular braided fabric on the outer layer of the C-PEEEUU-PEG-Hep/REDV nano fiber tubular stent to obtain a double-layer stent;
and step seven, assembling the double-layer stent together with the shaft core into a mold, and carrying out C-PEEEUU solution casting and treatment to obtain the artificial blood vessel.
Further, raw materials for preparing the composite spinning solution in the first step also comprise silk fibroin.
Further, the mass ratio of one or more master batches of PLGA, PDO and PGA to silk fibroin is 1: 1-4: 1.
further, the stainless steel is 316L stainless steel with the diameter of 1-6 mm.
Further, the thickness of the PEO nano-fiber spun on the stainless steel in the third step is 3-6 mu m, and the thickness of the C-PEEEUU-NH2The thickness of the nanofiber is 10-100 μm.
Further, degradable polyurethane elastomer C-PEEEUU-NH in the step three2The synthetic raw materials comprise PCL dihydric alcohol, N-Boc-serinol, hydroxyl-terminated PEG and HDI; wherein, PCL dihydric alcohol, N-Boc-serinol and terminal hydroxylMolar total amount of PEG: the molar amount of HDI was 1: 2.
further, the weight average molecular weight of the hydroxyl-terminated PEG is 500-1200.
Further, the C-PEEEUU-NH is treated in the fourth step2Grafting and modifying the nano-fiber tubular stent by using C-PEEEUU-NH2Soaking the nano-fiber tubular scaffold in ultrapure water, and vacuumizing and cleaning.
Further, step four H2The weight average molecular weight of the N-PEG-COOH is 1000-5000.
Further, the preparation method of the C-PEEEUU-PEG-Hep/REDV nano fiber tubular scaffold specifically comprises the following steps:
(1) the prepared C-PEEEUU-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(2) activating the above-mentioned H2Slowly dripping N-PEG-COOH activating solution into the solution containing C-PEEEUU-NH2In PBS buffer solution of the nano-fiber tubular scaffold, keeping out of the sun, sucking at room temperature for half an hour in a vacuum drying oven, and then gently oscillating for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally placing the mixture in a pre-freezing chamber at the temperature of minus 80 ℃, and then freezing and drying the mixture in a vacuum freezing dryer for 48 hours to obtain C-PEEEUU-PEG-NH2A nanofiber tubular scaffold;
(3) mixing the prepared C-PEEEUU-PEG-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(4) slowly dripping the activated heparin sodium/(Mpa) -GGGGGREDV polypeptide activating solution into solution containing C-PEEEUU-PEG-NH2In PBS buffer solution of nanofiber membrane, keeping out of the sunPumping for half an hour at room temperature in a vacuum drying box, and then gently shaking for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally, pre-freezing at-80 ℃, and freeze-drying for 48 hours to obtain the C-PEEEUU-PEG-Hep/REDV nano fiber tubular scaffold.
Further, the EDC/NHS activating solution is 0.05mol/L MES mixed buffer solution with pH 5.5 containing 0.06mol/L NHS, 0.12mol/L EDC and 0.5mol/L NaCl.
Further, in the step (4), the molar weight ratio of the heparin sodium to the (Mpa) -GGGGGREDV polypeptide in the heparin sodium/(Mpa) -GGGGGREDV polypeptide activation solution is 1: 1.
further, the specific steps of the seventh step are as follows:
sequentially sleeving the prepared C-PEEEUU-PEG-Hep/RGD nanofiber tubular scaffold and tubular braided fabric on a self-made polytetrafluoroethylene mold shaft core for phase separation, pouring a dissolved C-PEEEUU solution along the mold wall, covering a mold top cover, and freezing in a refrigerator at-80 ℃ for more than 3 hours; and (3) taking the pre-frozen pipe by using a foam ice box, opening a top cover of the mold, pinching the other end of the mold by hands, slowly drawing and adjusting a shaft core of the mold, putting the mold into ice water at 0 ℃ for exchanging the solvent DMSO, replacing the ice water every 6 hours, taking out the pipe in the ice water mixture after exchanging for 2 days, pre-freezing and freeze-drying to obtain the artificial blood vessel.
Further, the raw materials for synthesizing the degradable polyurethane elastomer C-PEEEUU in the step seven comprise PCL dihydric alcohol, hydroxyl-terminated PEG and HDI; wherein, the molar total amount of the PCL dihydric alcohol and the hydroxyl-terminated PEG is as follows: the molar amount of HDI was 1: 2.
the second aspect of the invention is to provide the braided reinforced degradable polyurethane elastomer artificial blood vessel prepared by the preparation method.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the spinning material C-PEEEUU-NH used in the invention2Has good promoting effect on adhesion, proliferation and migration of endothelial cells, and preparation method thereofThe preparation process is simple and stable, and the C-PEEEUU-NH grafted with PEG, heparin and (Mpa) -GGGGGREDV polypeptide2The nano-fiber is more beneficial to effective anticoagulation and platelet adhesion in the early stage of the stent and promotes endothelial cells to quickly overgrow the artificial vascular stent in the later stage, thereby realizing quick endothelialization, and simultaneously C-PEEEUU-NH2The nano fiber has adjustable mechanical property and plays a role in supporting the endothelial layer; the nanofiber yarns in the middle layer are woven into an enhancement layer to serve as an overall mechanical support layer of the tubular support, so that mechanical support is achieved in the early stage, larger holes are formed through later-stage degradation, and smooth muscle immigration is facilitated; the outer layer is the raw material C-PEEEUU prepared by thermally induced phase separation, the synthesis process is simple and stable, the synthesized molecular weight is controllable, and the outer layer of the C-PEEEUU prepared by thermally induced phase separation technology has communicated macropores, so that smooth muscle can be guided and promoted to grow quickly, and the function of remodeling the vascular wall is achieved. The preparation of the composite tissue engineering blood vessel with good tissue compatibility and biomechanical property provides a simple and effective preparation technical thought for developing small-caliber blood vessel stents.
The woven reinforced degradable polyurethane elastomer artificial blood vessel provided by the invention can be used for tissue regeneration and construction of cardiovascular and cerebrovascular diseases, the preparation method is simple and efficient, the price is low, the prepared tubular scaffold has good mechanical properties, bursting strength and biocompatibility, has no hypersensitivity to human body during transplantation operation, has affinity with blood and peripheral tissues in human body, and has good application prospect in intracardiac, surgical and brain surgical repair operations.
Drawings
FIG. 1 is a schematic view of a process for preparing a woven reinforced degradable polyurethane elastomer artificial blood vessel according to an embodiment of the present invention;
FIG. 2 is a scanning electron microscope image of the relevant products in the process of preparing the artificial blood vessel made of the reinforced woven degradable polyurethane elastomer according to one embodiment of the present invention; wherein A is a scanning electron microscope image of the spun PLGA/silk fibroin nano-fiber yarn; b is a scanning electron microscope image of the cross section of the prepared woven enhanced degradable polyurethane elastomer artificial blood vessel, wherein a is a C-PEEEUU-PEG-Hep/RGD nanofiber layer, B is a PLGA/silk fibroin nanofiber yarn woven layer, and C is a C-PEEEUU thermally induced phase separation macroporous layer; c is C-PEEEUU-PEG-Hep/RGD nano fiber; d is thermally phase separated C-PEEEUU.
FIG. 3 shows the results of biocompatibility and mechanical properties of the artificial blood vessel made of the reinforced and woven degradable polyurethane elastomer according to one embodiment of the present invention; wherein A is the proliferation condition of human umbilical vein endothelial cells after being co-cultured with TCP, coverlips and artificial blood vessels respectively; b is a radial tensile stress-strain curve of the prepared woven reinforced degradable polyurethane elastomer artificial blood vessel; c and D are respectively the compliance and bursting strength data of the prepared woven enhanced degradable polyurethane elastomer artificial blood vessel and the human mammary artery and the saphenous vein.
Detailed Description
The invention provides a preparation method of a woven reinforced degradable polyurethane elastomer artificial blood vessel, which comprises the following steps:
mixing and dissolving one or more master batches of PLGA, PDO and PGA to obtain a composite spinning solution;
spinning the composite spinning solution by adopting a double-needle-head opposite-spraying electrostatic spinning system to obtain nanofiber yarns, and weaving the nanofiber yarns into tubular braided fabrics;
spinning PEO (polyethylene oxide) nano-fibers on the stainless steel of the die by an electrostatic spinning technology, and then spinning C-PEEEUU-NH2A nanofiber; loading the PEO and C-PEEEUU-NH on the carrier2Putting the stainless steel of the nano-fiber into ultrapure water for demolding; removing the removed C-PEEEUU-NH2Continuously stirring and cleaning the nanofiber tubular support in ultrapure water until impurities on the surface of the nanofiber tubular support are removed, and then carrying out vacuum freeze drying;
step four, adopting activated H2N-PEG-COOH, heparin and (Mpa) -GGGGGREDV polypeptide on the C-PEEEUU-NH2The nano fiber tubular scaffold is obtained by grafting modification
C-PEEEUU-PEG-Hep/REDV nanofiber tubular scaffold;
step five, sheathing the shaft core of a special mould with the C-PEEEUU-PEG-Hep/REDV nano fiber tubular bracket, and then drying;
step six, sleeving the tubular braided fabric on the outer layer of the C-PEEEUU-PEG-Hep/REDV nano fiber tubular stent to obtain a double-layer stent;
and step seven, assembling the double-layer stent together with the shaft core into a mold, and carrying out C-PEEEUU solution casting and treatment to obtain the artificial blood vessel.
In a preferred embodiment of the present invention, the raw material for preparing the composite spinning solution in the first step further comprises silk fibroin.
In a preferred embodiment of the present invention, the mass ratio of one or more of PLGA, PDO, and PGA to silk fibroin is 1: 1-4: 1.
in a preferred embodiment of the present invention, the solvent used for dissolution in step one is THF: a mixed solvent of DMF (the volume ratio is 3: 1-5: 1), and the mass/volume concentration of the prepared mixed solution is 8-12%.
In a preferred embodiment of the present invention, the diameter distribution of the nanofibers constituting the nano yarn in the first step is 500 to 800 nm.
In a preferred embodiment of the present invention, the yarn for preparing the tubular braided fabric is one of PLGA yarn (12 PLGA monofilaments with a diameter of 10 μm are twisted into one braided yarn), PDO yarn (12 PDO monofilaments with a diameter of 10 μm are twisted into one braided yarn), PGA yarn (12 PDO monofilaments with a diameter of 10 μm are twisted into one braided yarn), PLGA/silk fibroin nanofiber yarn, PDO/silk fibroin nanofiber yarn, PLGA/PGA/silk fibroin nanofiber yarn, PGA/PDO/silk fibroin nanofiber yarn, PLGA/PDO/PGA/silk fibroin nanofiber yarn.
In a preferred embodiment of the present invention, the stainless steel is 316L stainless steel with a diameter of 1-6 mm.
In a preferred embodiment of the present invention, the thickness of the PEO nano-fiber spun on the stainless steel in the third step is 3-6 μm, C-PEEEUU-NH2The thickness of the nanofiber is 10-100 μm.
In a preferred embodiment of the present invention, the degradable polyurethane elastomer C-PEEEUU-NH in step three2The concrete steps of synthesisThe method comprises the following steps: mixing the monomers in a molar ratio of 1: 1: adding the PCL dihydric alcohol, the N-Boc serinol and the hydroxyl-terminated PEG of the 1 into a three-neck flask, and vacuumizing an oil pump to remove water for 3 hours at the temperature of 120 ℃ in an oil bath; under the protection of nitrogen, adding a certain amount of anhydrous DMSO to ensure that the mass volume concentration of the solution is 10%, adding HDI (the molar total amount of PCL dihydric alcohol, N-Boc-serinol and hydroxyl-terminated PEG: the molar amount of HDI is 1: 2) under the protection of nitrogen, and carrying out prepolymerization reaction for 3 hours at 80 ℃; then, the prepolymerization reaction solution was cooled to room temperature, and a DMSO solution containing 1, 4-butanediamine (mass volume concentration: 2%, molar amount of 1, 4-butanediamine: molar amount of HDI: 1: 2) was added dropwise to conduct a chain extension reaction, followed by a reaction at 40 ℃ for 18 hours. After the reaction, the final reaction solution was continuously added dropwise to a beaker of ultrapure water with a 20mL syringe for precipitation, and then the precipitate was purified by immersion in isopropanol for 24 hours, and vacuum-dried at 60 ℃ for 48 hours to obtain a white noodle-like solid product of C-PEEUU-Boc, the yield of which was calculated to be about 92%. Dissolving C-PEUU-Boc in chloroform/TFA (50/50) mixed solvent at concentration of 5% (w/v), deprotecting at room temperature for 24 hr, removing a large amount of chloroform and TFA solvent by rotary distillation, and reprecipitating on Na2CO3Residual TFA was removed by neutralization with aqueous solution (2% w/v, pH 11.4). Sucking the product with 20mL medical syringe, squeezing into deionized water solution, cleaning, soaking in isopropanol, purifying for 24 hr, vacuum drying at 60 deg.C for 48 hr to obtain white noodle-shaped C-PEEEUU-NH2Solid product, yield about 92%;
in the reaction, the monomer molar ratio of PCL diol, N-Boc serinol, hydroxyl-terminated PEG, HDI and putrescine is strictly controlled to be 0.1: 0.1: 0.1: 0.6: 0.3.
in a preferred embodiment of the present invention, the weight average molecular weight of the hydroxyl-terminated PEG in step three is 500-1200.
In a preferred embodiment of the invention, the mold is a polytetrafluoroethylene mold.
In a preferred embodiment of the present invention, C-PEEEUU-NH is para-reacted in step four2Grafting and modifying the nano-fiber tubular stent by using C-PEEEUU-NH2Soaking the nano-fiber tubular scaffold in ultrapure water, and vacuumizing and cleaning.
In a preferred embodiment of the inventionMiddle, step four middle H2The weight average molecular weight of the N-PEG-COOH is 1000-5000.
In a preferred embodiment of the present invention, the specific preparation method of the C-PEEEUU-PEG-Hep/REDV nanofiber tubular scaffold comprises the following steps:
(1) the prepared C-PEEEUU-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(2) activating the above-mentioned H2Slowly dripping N-PEG-COOH activating solution into the solution containing C-PEEEUU-NH2In PBS buffer solution of the nano-fiber tubular scaffold, keeping out of the sun, sucking at room temperature for half an hour in a vacuum drying oven, and then gently oscillating for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally placing the mixture in a pre-freezing chamber at the temperature of minus 80 ℃, and then freezing and drying the mixture in a vacuum freezing dryer for 48 hours to obtain C-PEEEUU-PEG-NH2A nanofiber tubular scaffold;
(3) mixing the prepared C-PEEEUU-PEG-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(4) slowly dripping the activated heparin sodium/(Mpa) -GGGGGREDV polypeptide activating solution into solution containing C-PEEEUU-PEG-NH2In PBS buffer solution of the nanofiber membrane, keeping out of the sun, sucking at room temperature for half an hour in a vacuum drying oven, and then gently oscillating for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally, pre-freezing at-80 ℃, and freeze-drying for 48 hours to obtain the C-PEEEUU-PEG-Hep/REDV nano fiber tubular scaffold.
In a preferred embodiment of the present invention, the structural formula of the (Mpa) -GGGGGREDV polypeptide is shown in formula 1:
in a preferred embodiment of the present invention, the EDC/NHS activating solution is a 0.05mol/L MES mixed buffer solution with pH 5.5 containing 0.06mol/L NHS, 0.12mol/L EDC and 0.5mol/L NaCl.
In a preferred embodiment of the present invention, in the step (4), the molar weight ratio of the heparin sodium in the heparin sodium/(Mpa) -GGGGGREDV polypeptide activation solution to the (Mpa) -GGGGGREDV polypeptide is 1: 1.
in a preferred embodiment of the present invention, the specific steps of step seven are as follows:
sequentially sleeving the prepared C-PEEEUU-PEG-Hep/RGD nanofiber tubular scaffold and tubular braided fabric on a self-made polytetrafluoroethylene mold shaft core for phase separation, pouring a dissolved C-PEEEUU solution along the mold wall, covering a mold top cover, and freezing in a refrigerator at-80 ℃ for more than 3 hours; and (3) taking the pre-frozen pipe by using a foam ice box, opening a top cover of the mold, pinching the other end of the mold by hands, slowly drawing and adjusting a shaft core of the mold, putting the mold into ice water at 0 ℃ for exchanging the solvent DMSO, replacing the ice water every 6 hours, taking out the pipe in the ice water mixture after exchanging for 2 days, pre-freezing and freeze-drying to obtain the artificial blood vessel.
In a preferred embodiment of the present invention, the degradable polyurethane elastomer C-peueu in step seven comprises the following specific synthesis steps: mixing the monomers in a molar ratio of 1: adding the PCL dihydric alcohol and the hydroxyl-terminated PEG of the 1 into a three-neck flask, and performing oil pump vacuum pumping to remove water for 3 hours at the temperature of 120 ℃ in an oil bath; under the protection of nitrogen, adding a certain amount of anhydrous DMSO to ensure that the mass volume concentration of the solution is 10%, adding HDI (the molar total amount of PCL dihydric alcohol and hydroxyl-terminated PEG: the molar amount of HDI is 1: 2) under the protection of nitrogen, and carrying out prepolymerization reaction for 3 hours at 80 ℃; then, the prepolymerization reaction solution was cooled to room temperature, and a DMSO solution containing 1, 4-butanediamine (mass volume concentration: 2%, molar amount of 1, 4-butanediamine: molar amount of HDI: 1: 2) was added dropwise to conduct a chain extension reaction, followed by a reaction at 40 ℃ for 18 hours. After the reaction, the final reaction solution was continuously added dropwise to a beaker of ultrapure water with a 20mL syringe for precipitation, and then the precipitate was purified by immersion in isopropanol for 24 hours, and vacuum-dried at 60 ℃ for 48 hours to obtain a white noodle-like solid product of C-PEEUU-Boc, the yield of which was calculated to be about 96%. In the reaction, the monomer molar ratio of PCL diol, hydroxyl-terminated PEG, HDI and putrescine is strictly controlled to be 0.15: 0.15: 0.6: 0.3.
the present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
The embodiment provides a woven reinforced degradable polyurethane elastomer artificial blood vessel, and referring to fig. 1 and fig. 2, the preparation method comprises the following steps:
(1) weaving PLGA/silk fibroin nanofiber yarns (the mass ratio of PLGA to silk fibroin is 1: 1) (12 yarns with the monofilament diameter of 10 mu m in total) into a tubular braided fabric;
(2) spinning PEO nano-fibers with the thickness of about 5 μm on 316L stainless steel with the diameter of 2mm by an electrostatic spinning technology; spinning on the fiber to a thickness of 100. mu.mC-PEEEUU-NH2A nanofiber; the above-mentioned polymer is connected with PEO and C-PEEEUU-NH2Putting 316L stainless steel of the nanofiber into ultrapure water for demolding; removing the removed C-PEEEUU-NH2Continuously stirring and cleaning the nanofiber tubular scaffold in ultrapure water for 30min to remove impurities on the surface of the fiber, and then carrying out vacuum freeze drying for later use;
(3) the prepared C-PEEEUU-NH2Soaking the nanofiber tubular scaffold in ultrapure water, vacuum-cleaning for 5 times, and subjecting the activated graft (H)2N-PEG2kGrafting modification is carried out on-COOH, heparin and (Mpa) -GGGGGREDV polypeptide in sequence; finally, obtaining the functionalized C-PEEEUU-PEG2k-Hep/REDV nanofiber tubular scaffold;
(4) sheathing the shaft core of the mold with C-PEEEUU-PEG2k-placing the Hep/REDV nano fiber tubular scaffold, the nano fiber and the shaft core into a vacuum drying oven for drying;
(5) sleeving the obtained tubular braided fabric on the outer layer sleeved with the nanofiber shaft core to serve as a mechanical enhancement layer;
(6) and assembling the composite double-layer stent together with the shaft core into a mold, and carrying out C-PEEEUU solution casting and treatment to obtain the target artificial blood vessel.
Example 2
The embodiment provides a woven reinforced degradable polyurethane elastomer artificial blood vessel, and referring to fig. 1 and fig. 3, the preparation method comprises the following steps:
(1) weaving PLGA/PDO/PGA/silk fibroin nanofiber yarns (the mass ratio of the total mass of PLGA/PDO/PGA to the mass of silk fibroin is 4: 1, wherein the mass ratio of PLGA, PDO and PGA in the total mass of PLGA/PDO/PGA is 1: 1: 1) (12 yarns in total with the monofilament diameter of 10 mu m) into a tubular braided fabric;
(2) spinning PEO nano-fibers with the thickness of about 5 μm on 316L stainless steel with the diameter of 2mm by an electrostatic spinning technology; spinning on the fiber to a thickness of 100. mu.mC-PEEEUU-NH2A nanofiber; the above-mentioned polymer is connected with PEO and C-PEEEUU-NH2Putting 316L stainless steel of the nanofiber into ultrapure water for demolding; removing the removed C-PEEEUU-NH2Continuously stirring and cleaning the nanofiber tubular scaffold in ultrapure water for 30min to remove impurities on the surface of the fiber, and then carrying out vacuum freeze drying for later use;
(3) the prepared C-PEEEUU-NH2Soaking the nanofiber tubular scaffold in ultrapure water, vacuum-cleaning for 5 times, and subjecting the activated graft (H)2N-PEG2kGrafting modification is carried out on-COOH, heparin and (Mpa) -GGGGGREDV polypeptide in sequence; finally, obtaining the functionalized C-PEEEUU-PEG2k-Hep/REDV nanofiber tubular scaffold;
(4) sheathing the shaft core of the mold with C-PEEEUU-PEG2k-placing the Hep/REDV nano fiber tubular scaffold, the nano fiber and the shaft core into a vacuum drying oven for drying;
(5) sleeving the obtained tubular braided fabric on the outer layer sleeved with the nanofiber shaft core to serve as a mechanical enhancement layer;
(6) and assembling the composite double-layer stent together with the shaft core into a mold, and carrying out C-PEEEUU solution casting and treatment to obtain the target artificial blood vessel.
As can be seen from FIG. 3, the artificial blood vessel has safe cell compatibility, no cell contact death phenomenon, reliable and safe biomechanics and mechanical compliance and bursting strength superior to those of human milk arteries and saphenous veins.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (12)
1. A preparation method of a woven reinforced degradable polyurethane elastomer artificial blood vessel is characterized by comprising the following steps:
mixing and dissolving one or more master batches of PLGA, PDO and PGA to obtain a composite spinning solution;
spinning the composite spinning solution by adopting a double-needle-head opposite-spraying electrostatic spinning system to obtain nanofiber yarns, and weaving the nanofiber yarns into tubular braided fabrics;
spinning PEO nano-fiber on the stainless steel of the die by an electrostatic spinning technology, and then spinning C-PEEEUU-NH2A nanofiber; loading PEO and C-PEEEUU-NH2Putting the stainless steel of the nano-fiber into ultrapure water for demolding; removing the removed C-PEEEUU-NH2Continuously stirring and cleaning the nanofiber tubular support in ultrapure water until impurities on the surface of the nanofiber tubular support are removed, and then carrying out vacuum freeze drying;
step four, adopting activated H2N-PEG-COOH, heparin and (Mpa) -GGGGGREDV polypeptide pair C-PEEEUU-NH2Grafting and modifying the nano fiber tubular scaffold to obtain C-PEEEUU-PEG-Hep/REDV sodiumA rice fiber tubular scaffold;
step five, sleeving the shaft core of a special mould on the C-PEEEUU-PEG-Hep/REDV nano fiber tubular bracket, and then drying;
step six, sleeving the tubular braided fabric on the outer layer of the C-PEEEUU-PEG-Hep/REDV nano fiber tubular stent to obtain a double-layer stent;
and step seven, assembling the double-layer stent together with the shaft core into a mold, and carrying out C-PEEEUU solution casting and treatment to obtain the artificial blood vessel.
2. The method according to claim 1, wherein raw materials for preparing the composite spinning solution in the first step further comprise silk fibroin.
3. The preparation method according to claim 2, wherein the mass ratio of one or more master batches of PLGA, PDO and PGA to silk fibroin is 1: 1-4: 1.
4. the method of claim 1, wherein the thickness of the PEO nanofibers spun on the stainless steel in step three is 3-6 μm, C-PEEEUU-NH2The thickness of the nanofiber is 10-100 μm.
5. The method of claim 1, wherein the C-PEEEUU-NH is present in step III2The synthetic raw materials comprise PCL dihydric alcohol, N-Boc-serinol, hydroxyl-terminated PEG and HDI; wherein, the molar total amount of PCL dihydric alcohol, N-Boc-serinol and hydroxyl-terminated PEG is as follows: the molar amount of HDI was 1: 2.
6. the method according to claim 5, wherein the weight average molecular weight of the hydroxyl terminated PEG is 500 to 1200.
7. The process according to claim 1, wherein H is the product of step four2The weight average molecular weight of the N-PEG-COOH is 1000-5000.
8. The method for preparing the tubular scaffold according to claim 1, wherein the C-PEEEUU-PEG-Hep/REDV nanofiber scaffold is prepared by a specific method comprising the following steps:
(1) the prepared C-PEEEUU-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(2) activating the activated H2Slowly dripping N-PEG-COOH activating solution into the solution containing C-PEEEUU-NH2In PBS buffer solution of the nano-fiber tubular scaffold, keeping out of the sun, sucking at room temperature for half an hour in a vacuum drying oven, and then gently oscillating for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally placing the mixture in a pre-freezing chamber at the temperature of minus 80 ℃, and then freezing and drying the mixture in a vacuum freezing dryer for 48 hours to obtain C-PEEEUU-PEG-NH2A nanofiber tubular scaffold;
(3) mixing the prepared C-PEEEUU-PEG-NH2Soaking the nano-fiber tubular bracket in newly prepared 0.05mol/L MES solution, and magnetically stirring for 30 minutes; soaking the nanofiber membrane or the nanofiber tubular scaffold in EDC/NHS activating solution, keeping out of the sun, sucking at room temperature in a vacuum drying box for half an hour, and then oscillating for 3 hours by a shaking table;
(4) slowly dripping the activated heparin sodium/(Mpa) -GGGGGREDV polypeptide activating solution into solution containing C-PEEEUU-PEG-NH2In PBS buffer solution of the nanofiber membrane, keeping out of the sun, sucking at room temperature for half an hour in a vacuum drying oven, and then gently oscillating for 48 hours; after the grafting reaction is finished, taking out the nanofiber membrane or the nanofiber tubular scaffold, carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using PBS (Poly Butylene succinate), and carrying out magnetic stirring and cleaning for 3 times and 10 minutes each time by using ultrapure water; finally, pre-freezing at-80 ℃, and freeze-drying for 48 hours to obtain the C-PEEEUU-PEG-Hep/REDV nano fiber tubular scaffold.
9. The method of claim 8, wherein the EDC/NHS activating solution is 0.05mol/L MES mixed buffer solution with pH 5.5 containing 0.06mol/L NHS, 0.12mol/L EDC, and 0.5mol/L NaCl.
10. The method according to claim 8, wherein the molar ratio of heparin sodium to the (Mpa) -GGGGGREDV polypeptide in the heparin sodium/(Mpa) -GGGGGREDV polypeptide activation solution in the step (4) is 1: 1.
11. the method according to claim 1, wherein the raw materials for the synthesis of C-peueu in step seven comprise PCL diol, hydroxyl terminated PEG, and HDI; wherein, the molar total amount of the PCL dihydric alcohol and the hydroxyl-terminated PEG is as follows: the molar amount of HDI was 1: 2.
12. a woven reinforced degradable polyurethane elastomer artificial blood vessel prepared by the preparation method of any one of claims 1 to 11.
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