CN116173300B - Plant decellularized artificial blood vessel and preparation method thereof - Google Patents

Plant decellularized artificial blood vessel and preparation method thereof Download PDF

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CN116173300B
CN116173300B CN202211454496.0A CN202211454496A CN116173300B CN 116173300 B CN116173300 B CN 116173300B CN 202211454496 A CN202211454496 A CN 202211454496A CN 116173300 B CN116173300 B CN 116173300B
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decellularized
plant
blood vessel
hydrogel
exosome
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CN116173300A (en
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张金盈
侯雅尘
唐俊楠
沈德良
崔小林
张格�
秦臻
郭嘉城
曹昶
苏畅
杜鹏翀
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First Affiliated Hospital of Zhengzhou University
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3637—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/507—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials for artificial blood vessels
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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Abstract

The invention belongs to the technical field of preparation of artificial blood vessels, and particularly relates to a plant decellularized artificial blood vessel and a preparation method thereof. The preparation method provided by the invention comprises the following steps: the plant roots and/or stems with the hollow structure are decellularized to obtain a decellularized scaffold; coating the photopolymerized hydrogel-exosome suspension on the outer surface of the decellularized scaffold under the condition of illumination with the wavelength of 365-550 nm for photocuring reaction to obtain the plant decellularized artificial blood vessel; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator. The plant decellularized artificial blood vessel prepared by the invention can promote the adhesion and proliferation of endothelial cells and imitate the natural form of the human blood vessel media so as to obtain the artificial blood vessel with good bionic and endothelialization effects. The preparation method provided by the invention has the advantages of simple flow, no need of expensive reagents, low cost and good endothelialization effect of the prepared artificial blood vessel.

Description

Plant decellularized artificial blood vessel and preparation method thereof
Technical Field
The invention belongs to the technical field of preparation of artificial blood vessels, and particularly relates to a plant decellularized artificial blood vessel and a preparation method thereof.
Background
Cardiovascular disease (CVD) is a major cause of global morbidity and mortality, with an estimated annual death of about 1780 tens of thousands (233.1 out of every 10 tens of thousands) from CVD. CVD is routinely treated by vascular bypass surgery using vascular grafts to bypass blood flow around the occlusion site. In the vascular bypass surgery, the use of autologous blood vessels is a gold standard, but the prevalence of autologous blood vessels is often limited due to underlying diseases that may be present in the patient.
Currently, commercially available artificial grafts are made of polymers, either expanded polytetrafluoroethylene (ePTFE; known as Gore-Tex) or polyethylene terephthalate (PET; known as Dacron), both of which can be prepared into a variety of large caliber (> 6 mm) vessels for storage. However, the polymer blood vessel is hard, has a rough surface, and has a highly hydrophobic surface, resulting in poor biocompatibility of the blood vessel and easy activation of cascade reaction of blood coagulation, so that the polymer is not suitable for small-caliber artificial blood vessels. Despite the current availability of 3D printing technology and microfluidic technology, there are still significant challenges in replicating microvascular structures.
Disclosure of Invention
The invention aims to provide a plant acellular artificial blood vessel and a preparation method thereof, and the plant acellular artificial blood vessel provided by the invention is suitable for small-caliber artificial blood vessels and has good bionic and endothelialization effects.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a plant decellularized artificial blood vessel, which comprises the following steps:
the plant roots and/or stems with the hollow structure are decellularized to obtain a decellularized scaffold;
coating the photopolymerized hydrogel-exosome suspension on the outer surface of the acellular bracket under the condition of illumination to carry out a photocuring reaction so as to obtain the plant acellular artificial blood vessel; the wavelength of the illumination is 365-550 nm; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator.
Preferably, the photopolymerizable hydrogel in the photopolymerizable hydrogel solution comprises one or more of methacrylic anhydride gelatin, methacryloylated hyaluronic acid, methacryloylated collagen, tyramine substituted hyaluronic acid, tyramine collagen and tyramine gelatin.
Preferably, the exosome source comprises one or more of blood serum, bone marrow mesenchymal stem cells, adipose stem cells, vascular endothelial cell sources, and vascular contractile smooth muscle cell sources.
Preferably, the mass percentage of the photopolymerization hydrogel contained in the photopolymerization hydrogel solution is 5-10wt%.
Preferably, in the photopolymerized hydrogel-exosome suspension, the mass concentration of the exosome source is 2-200 mug/mu L.
Preferably, the photoinitiator comprises one or more of Irgacure2959, VA-086, eosin Y and Ru/SPS; in the photopolymerized hydrogel-exosome suspension, the mass percentage of the photoinitiator is 0.1-1 wt%.
Preferably, the coating is spray coating, and the working parameters of the spray coating include: the rotation speed of the decellularized scaffold is 50-200 r/min; the speed of the photopolymerized hydrogel-exosome suspension sprayed out by the spray head is 0.1-2 mu L/s.
Preferably, the spraying is performed intermittently, and the duration of each spraying is 1-3 s; the interval time between two adjacent spraying is 5-60 s.
Preferably, the decellularization comprises the steps of:
using a glass rod to penetrate through a hollow part of a plant root and/or stem with a hollow structure as a support, and then immersing the plant root and/or stem with the hollow structure in a hexane solution to remove horny layers on the surface of the plant root and/or stem, thereby obtaining exfoliating roots and/or stems;
pouring sodium dodecyl sulfate solution into the hollow structure of the exfoliating root and/or stem to perform pretreatment for removing plant cell tissues, so as to obtain pretreated root and/or stem;
washing the outer surface of the pretreatment root and/or stem and the inner surface of the hollow structure by using a mixed solution of polyethylene glycol octyl phenyl ether and sodium chlorite, and carrying out treatment for removing plant cell tissues to obtain the decellularized scaffold.
The invention provides a plant acellular artificial blood vessel prepared by the preparation method of the technical scheme, which comprises an acellular bracket and a hydrogel-exosome photocuring coating attached to the acellular bracket.
The invention provides a preparation method of a plant decellularized artificial blood vessel, which comprises the following steps: the plant roots and/or stems with the hollow structure are decellularized to obtain a decellularized scaffold; coating the photopolymerized hydrogel-exosome suspension on the outer surface of the acellular bracket under the condition of illumination to carry out a photocuring reaction so as to obtain the plant acellular artificial blood vessel; the wavelength of the illumination is 365-550 nm; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator. The invention carries out decellularization on plant roots and/or stems with a hollow structure to obtain a decellularized scaffold, further coats the decellularized scaffold with photopolymerization hydrogel-exosome suspension, rapidly and uniformly solidifies the hydrogel on the surface of the decellularized scaffold under the illumination condition with the wavelength of 365-550 nm, wraps the exosome, maintains the exosome in vitro activity by means of the bionic environment of the hydrogel, and forms a stable artificial vascular structure, thus being suitable for small-caliber artificial blood vessels; the coating film structure of the solidified hydrogel coated exosome constructed on the surface of the artificial blood vessel wall can promote the adhesion and proliferation of endothelial cells and imitate the natural form of the human blood vessel medium film so as to obtain the artificial blood vessel with good bionic and endothelialization effects. The preparation method provided by the invention has the advantages of simple flow, no need of expensive reagents, low cost and good endothelialization effect of the prepared artificial blood vessel.
The invention provides a plant acellular artificial blood vessel prepared by the preparation method, which comprises an acellular bracket and a hydrogel-exosome photocuring coating attached to the acellular bracket. The invention provides good supporting capability for the artificial blood vessel through the hollow tubular fiber structure, and is suitable for the small-caliber artificial blood vessel; the hydrogel-exosome coating after photocuring imitates the membranous structure in the human blood vessel, the hydrogel after curing has rich three-dimensional reticular pore structures, and can promote the adhesion and proliferation of endothelial cells while wrapping exosomes, thereby having good bionic and endothelialization effects.
Drawings
FIG. 1 is a schematic diagram of an apparatus for photocuring reaction of a decellularized scaffold coated photopolymerizable hydrogel-exosome suspension according to an embodiment of the invention;
in fig. 1, 1 is a supporting table, 2 is a rotating clamp, 3 is a rotating shaft, 4 is a decellularized scaffold, 5 is a nozzle, 6 is a raw material storage container, and 7 is a light source;
FIG. 2 is a schematic illustration of the decellularized scaffold of example 2 of the present invention;
FIG. 3 is a schematic illustration of a Glema-VSMC decellularized vascular prosthesis prepared in accordance with example 2 of the present invention;
FIG. 4 is an electron micrograph of a Glema-VSMC decellularized vascular prosthesis prepared in example 3 taken by a cryo-electron microscope;
FIG. 5 is a stress-strain curve of Glema used in example 4 of the present invention;
FIG. 6 is a stress-strain curve of the decellularized scaffold prepared in accordance with example 4 of the present invention;
FIG. 7 is a stress-strain curve of a Glema-VSMC-decellularized vascular prosthesis prepared in accordance with example 4 of the present invention;
FIG. 8 shows DPAI photo-staining of nuclei after 1 day co-culture of Glema-VSMC decellularized vascular prosthesis extract prepared in example 5 of the present invention with endothelial cells;
Detailed Description
The invention provides a preparation method of a plant decellularized artificial blood vessel, which comprises the following steps:
the plant roots and/or stems with the hollow structure are decellularized to obtain a decellularized scaffold;
coating the photopolymerized hydrogel-exosome suspension on the outer surface of the acellular bracket under the condition of illumination to carry out a photocuring reaction so as to obtain the plant acellular artificial blood vessel; the method comprises the steps of carrying out a first treatment on the surface of the The wavelength of the illumination is 365-550 nm; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
The invention carries out decellularization on plant roots and/or stems with hollow structures to obtain the decellularized scaffold.
In the present invention, the plant root and/or stem is preferably the root and/or stem of water spinach.
In the present invention, the inner diameter of the plant root and/or stem is preferably 1 to 3mm; the length of the plant root and/or stem is preferably 1.5-5 cm.
In the present invention, the decellularization preferably includes the steps of:
using a glass rod to penetrate through a hollow part of a plant root and/or stem with a hollow structure as a support, and then immersing the plant root and/or stem with the hollow structure in a hexane solution to remove horny layers on the surface of the plant root and/or stem, thereby obtaining exfoliating roots and/or stems;
pouring sodium dodecyl sulfate solution into the hollow structure of the exfoliating root and/or stem to perform pretreatment for removing plant cell tissues, so as to obtain pretreated root and/or stem;
washing the outer surface of the pretreatment root and/or stem and the inner surface of the hollow structure by using a mixed solution of polyethylene glycol octyl phenyl ether and sodium chlorite, and carrying out treatment for removing plant cell tissues to obtain the decellularized scaffold.
The invention uses a glass rod material to pass through the hollow part of the plant root and/or stem with a hollow structure as a support, and then the plant root and/or stem with the hollow structure is immersed in a hexane solution to remove the cuticle on the surface of the plant root and/or stem, thus obtaining the exfoliating root and/or stem.
The plant root and/or stem having a hollow structure is preferably subjected to a pretreatment before being immersed in a hexane solution, and in the present invention, the pretreatment preferably includes: immersing the plant roots and/or stems with the hollow structure in water for washing. In the present invention, the temperature of the immersion washing is preferably 4 ℃; the water is preferably ultrapure water, and the time for the immersion washing is preferably 2 to 6 hours. The present invention preferably removes stains from the surface of the plant roots and/or stems by the dip-wash.
In the present invention, the hexane solution is particularly preferably a phosphate buffer solution (PBS buffer solution) of hexane.
In the present invention, the mass percentage of the hexane solution is preferably 90wt%.
In the present invention, the amount of the hexane solution is particularly preferably 5 to 10mL.
In the present invention, the temperature of the hexane solution impregnation is preferably room temperature; the time for the hexane solution impregnation is preferably 4 to 24 hours.
In the present invention, after the completion of the impregnation with the hexane solution, the present invention preferably washes the exfoliating roots and/or stems, and the washing solvent is preferably a PBS buffer solution; the washing is preferably rinsing; the number of times of washing is preferably 3; the present invention preferably removes hexane and cuticle residue remaining on the exfoliating root and/or stem surface by washing.
After the exfoliating root and/or stem is obtained, the invention pours sodium dodecyl sulfate solution into the hollow structure of the exfoliating root and/or stem to perform the pretreatment of plant cell tissue, thus obtaining the pretreated root and/or stem.
In the present invention, the Sodium Dodecyl Sulfate (SDS) solution is preferably a PBS buffer solution of SDS.
In the present invention, the mass percentage of the SDS solution is preferably 1 to 10wt%.
In the present invention, the pouring is preferably performed using a peristaltic pump, and the pouring speed of the peristaltic pump is preferably 20 to 200r/min.
In the present invention, the temperature of the pre-removal plant cell tissue treatment is preferably room temperature; the time for the pre-removal of plant cell tissue treatment is preferably 5 to 7 days.
In the present invention, after the pretreatment of the plant cell tissue is completed, the present invention preferably washes the pretreated roots and/or stems, and the washing solvent is preferably a PBS buffer solution; the washing is preferably rinsing; the number of times of washing is preferably 3; the present invention preferably removes SDS and cellular tissue residues remaining on the surface of the pretreated roots and/or stems by washing.
After the pretreatment root and/or stem is obtained, the invention washes the outer surface of the pretreatment root and/or stem and the inner surface of the hollow structure by using a mixed solution of polyethylene glycol octyl phenyl ether (Triton-X-100) and sodium chlorite, and carries out the treatment of removing the plant cell tissue to obtain the decellularized scaffold.
In the present invention, the mixed solution of Triton-X-100 and sodium chlorite is preferably PBS buffer solution of Triton-X-100 and sodium chlorite.
In the invention, the mass percentage of the Triton-X-100 in the mixed solution of the Triton-X-100 and sodium chlorite is preferably 0.1 to 0.5 percent.
In the invention, the mass percentage of sodium chlorite in the mixed solution of Triton-X-100 and sodium chlorite is preferably 10wt%.
In the present invention, the flushing is preferably performed using a peristaltic pump, and the speed of flushing by the peristaltic pump is preferably 20 to 200r/min.
In the present invention, the volume of the mixed solution of Triton-X-100 and sodium chlorite is preferably 5-10 mL.
In the present invention, the temperature of the flushing is preferably room temperature; the flushing time is preferably 12 to 144 hours. In the present invention, it is preferable that the mixed solution of Triton-X-100 and sodium chlorite is changed every 12 hours during the washing.
In the present invention, after the completion of the treatment for removing plant cell tissue, the present invention preferably washes the obtained decellularized scaffold. The washing solvent is preferably PBS buffer solution; the washing is preferably rinsing; the number of times of washing is preferably 3; the present invention is preferably carried out by washing Triton-X-100, sodium chlorite and cell tissue residues remaining on the surface of the decellularized scaffold.
After obtaining the decellularized scaffold, the invention preferably immerses the decellularized scaffold in deionized water for 24 hours.
The decellularized scaffold is preferably stored in sterile deionized water at 4 ℃.
After obtaining a decellularized scaffold, coating photopolymerized hydrogel-exosome suspension on the outer surface of the decellularized scaffold under the condition of illumination to perform a photocuring reaction to obtain the plant decellularized artificial blood vessel; the wavelength of the illumination is 365-550 nm; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator.
In the present invention, the photopolymerizable hydrogel in the photopolymerizable hydrogel solution preferably includes one or more of methacrylic anhydride-based gelatin (GelMA), methacryloyl hyaluronic acid (HAMA), methacryloyl collagen (ColMA), tyramine-substituted hyaluronic acid (hatir), tyramine collagen (ColTyr) and tyramine-based gelatin (GelTyr), more preferably GelMA.
In the present invention, the mass percentage of the photopolymerizable hydrogel contained in the photopolymerizable hydrogel solution is preferably 5 to 10wt%.
In the present invention, the exosome source preferably includes one or more of blood serum, bone marrow mesenchymal stem cells, adipose stem cells, vascular endothelial cell sources, and vascular contractile smooth muscle cell (VSMC) sources.
In the present invention, the method for culturing VSMC preferably comprises the steps of:
amplifying the primary human VSMC by using a VSMC culture medium, carrying out VSMC passage treatment after the VSMC density reaches 90%, and carrying out enzyme treatment on the VSMC passage culture solution by using pancreatin after the VSMC cell density reaches 90%, so as to obtain a VSMC suspension; and carrying out solid-liquid separation on the VSMC suspension to obtain the VSMC precipitate, and discarding the supernatant. In the present invention, the VSMC medium is preferably a VSMC-dedicated medium purchased from (icellbiosciences inc, shanghai); the human primary human VSMC is preferably purchased from (icellbiosciences inc, shanghai); the invention preferably uses 75T culture flask for the amplification and passage treatment; in the present invention, the VSMC suspension preferably has a cell concentration of 10 5 ～10 7 cell/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The solid-liquid separation is preferably centrifugation.
In the present invention, the mass concentration of the exosome source in the photopolymerized hydrogel-exosome suspension is preferably 2 to 200. Mu.g/. Mu.L.
In the present invention, the photoinitiator comprises one or more of Irgacure2959, VA-086, eosin Y and Ru/SPS, more preferably Ru/SPS.
In the present invention, the mass percentage of the photoinitiator in the photopolymerizable hydrogel-exosome suspension is preferably 0.1 to 1wt%, more preferably 0.1 to 0.8wt%.
In the present invention, the preparation method of the photopolymerized hydrogel-exosome suspension preferably comprises the following steps:
carrying out first mixing on the photopolymerized hydrogel and PBS buffer solution under the light-shielding condition to obtain photopolymerized hydrogel solution;
mixing the photopolymerized hydrogel solution with the exosome to obtain a second mixed solution;
and thirdly mixing the first mixed solution and the photoinitiator under the light-shielding condition to obtain the photopolymerized hydrogel-exosome suspension.
In the present invention, the first mixing is preferably performed under stirring conditions, the stirring is preferably performed using a magnetic stirrer, and the rotation speed of the stirring is preferably 200 to 1000r/min, more preferably 250 to 900r/min. In the present invention, the temperature of the first mixing is preferably 35 to 40 ℃; the stirring time for mixing is preferably 5 to 60 minutes.
In the present invention, the second mixed solution is preferably stored in a sterile environment protected from light and at 4 ℃.
In the present invention, the first mixed solution is preferably used within 1 hour.
In the present invention, the coating is preferably spray coating, and the operating parameters of the spray coating preferably include: the rotation speed of the decellularized scaffold is 50-200 r/min, more preferably 60-180 r/min; the speed of the photopolymerized hydrogel-exosome suspension sprayed out by the spray head is 0.1-2 mu L/s, more preferably 0.15-1.5 mu L/s.
In the present invention, the coating is preferably carried out in a spraying apparatus as shown in fig. 1; in fig. 1: 1 is a supporting table, 2 is a rotary clamp, 3 is a rotary shaft, 4 is a decellularized bracket, 5 is a nozzle, 6 is a raw material storage container, and 7 is a light source. In the present invention, the light source 7 is preferably fixedly attached to the inner bottom surface of the support table 1; the rotary clamp 2 is preferably fixedly connected to the inner side wall of the supporting table 1; the rotating shaft 3 is preferably connected with the rotating clamp 2 in a rotating way; the raw material storage container 6 is preferably in communication with the nozzle 5 through a pipe; the nozzle 5 is preferably located above the rotation shaft 3.
The hollow structure of the decellularized scaffold is preferably inserted into the rotating shaft 3, and the decellularized scaffold is rotated in the spraying process by rotating the fixture 2.
In the present invention, the spraying is preferably performed intermittently, and the duration of each spraying is preferably 1 to 3 seconds; the interval between two adjacent spraying is preferably 5 to 60 seconds, more preferably 10 to 30 seconds.
In the present invention, the number of intermittent times of the spraying is preferably 1 to 9.
The invention preferably adopts intermittent spraying, the mixed system of the photopolymerization hydrogel-exosome is stably sprayed on the surface of the decellularized scaffold in a layer-by-layer photocuring mode, and the hydrogel-exosome mixture is rapidly and uniformly cured on the surface of the decellularized scaffold by adding light energy, so that a stable artificial blood vessel structure is formed; the invention simulates the natural form of the human body blood vessel medium membrane by constructing the structure of the hydrogel-exosome after photo-curing on the surface of the artificial blood vessel wall, so as to obtain the artificial blood vessel with good bionic and endothelialization effects.
The invention provides a plant acellular artificial blood vessel prepared by the preparation method, which comprises an acellular bracket and a hydrogel-exosome photocuring coating attached to the acellular bracket.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Taking root and stem of water spinach with the length of 1.5cm and the inner diameter of 1mm, soaking in ultrapure water at the temperature of 4 ℃ for 4 hours to remove surface stains; passing a glass rod with a length of 3cm and a diameter of 1mm through the hollow part as a support, diluting hexane to 85wt% with PBS buffer solution to obtain hexane solution, soaking rhizome of water spinach with 10mL of hexane solution for 6h to remove cuticle on the plant surface, and washing the surface with PBS buffer solution for 3 times to remove hexane and cuticle residues; SDS was diluted to 5wt% with PBS buffer and perfused at the hollow site for 7 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.5wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted mixed solution of Triton-X-100 and sodium chlorite, wherein the mass percent of Triton-X-100 is 0.5wt% and the mass percent of sodium chlorite is 10wt%, flushing hollow parts of rhizomes and the outer part of plants by using the mixed solution of 5mLTriton-X-100 and sodium chlorite for 144 hours, replacing the new mixed solution of Triton-X-100 and sodium chlorite every 12 hours, and flushing the surface 3 times by using PBS buffer solution to remove Triton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 24h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: the mixture is stirred in a dark place by using PBS buffer solution according to the concentration of 5 weight percent, the rotating speed of a magnetic stirrer is 200r/min, the temperature is 37 ℃ and the time is 10min, and clear solution is obtained; human primary human VSMC (cells purchased from icellbiosciences inc, shanghai) were selected, expanded using 75T flasks using dedicated medium (purchased from icellbiosciences inc), and when cell density reached 90%, passaged using T75 flasks; after the cell number reached 90%, pancreatin was treated to give a concentration of 10 7 cell/cm 2 After obtaining cell sediment by using a centrifugal machine, discarding the supernatant; cell pellet was reconstituted to a concentration of 10 using Gelma solution 7 cell/cm 2 Is stored aseptically at 4 ℃ in the dark and used within 1 hour. Before use, 0.1wt% Ru/SPS is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in fig. 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 50r/min, a spray head is sprayed at a speed of 0.2 mu L/s, and continuous 500nm wave band light is matched for curing. After each spraying for 2s, stopping, enhancing the light curing for 15s, starting the spraying process again, and repeating the process 5 times. The obtained Glema-VSMC decellularized vascular prosthesis is soaked in PBS and stored at 4 ℃.
Example 2
Taking root and stem of water spinach with the length of 3cm and the inner diameter of 1.5mm, soaking in ultrapure water at the temperature of 4 ℃ for 6 hours to remove surface stains; a glass bar with the length of 5cm and the diameter of 1.5mm is used for penetrating through the hollow part to be used as a support, hexane is diluted to 90wt% by using PBS buffer solution to obtain a hexane solution, 8mL of hexane solution is used for soaking the rhizome of the water spinach for 4 hours to remove cuticle on the plant surface, and the surface is washed 3 times by using the PBS buffer solution to remove hexane and cuticle residues; SDS was diluted to 7wt% with PBS buffer and perfused at the hollow site for 5 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.5wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted mixed solution of Triton-X-100 and sodium chlorite, wherein the mass percent of Triton-X-100 is 0.5wt% and the mass percent of sodium chlorite is 10wt%, flushing hollow parts of rhizomes and the outside of plants by using the mixed solution of 10mLTriton-X-100 and sodium chlorite for 48 hours, replacing the new mixed solution of Triton-X-100 and sodium chlorite every 12 hours, and flushing the surface 3 times by using PBS buffer solution to remove Triton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 24h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: the mixture is stirred in a dark place by using PBS according to the concentration of 6 weight percent, the rotating speed of a magnetic stirrer is 400r/min, the temperature is 37 ℃ and the time is 15min, and a clear solution is obtained; human primary human VSMC (cells purchased from icellbiosciences inc, shanghai) were selected, expanded using 75T flasks using dedicated medium (purchased from icellbiosciences inc), and when cell density reached 90%, passaged using T75 flasks; after the cell number reached 90%, pancreatin was treated to give a concentration of 10 7 cell/cm 2 After obtaining cell sediment by using a centrifugal machine, discarding the supernatant; cell pellet was reconstituted to a concentration of 10 using Gelma solution 7 cell/cm 2 Is stored aseptically at 4 ℃ in the dark and used within 1 hour. Before use, 0.5wt% Ru/SPS is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in fig. 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 100r/min, a spray head is sprayed at a speed of 0.3 mu L/s, and continuous 530nm wave band light is matched for curing. After each spraying for 2s, stopping, enhancing the photo-curing for 20s, starting the spraying process again, and repeating the process 5 times in total. The obtained Glema-VSMC decellularized vascular prosthesis is soaked in PBS and stored at 4 ℃.
FIG. 2 is a schematic view of the decellularized scaffold prepared in this example; FIG. 3 shows the patency of the Glema-VSMC decellularized vascular prosthesis prepared in this example.
Example 3
Taking root and stem of water spinach with length of 2cm and inner diameter of 1mm, soaking in ultrapure water at 4 ℃ for 6 hours to remove surface stains; passing a glass rod with a length of 3cm and a diameter of 1mm through the hollow part as a support, diluting hexane to 90wt% with PBS buffer solution to obtain hexane solution, soaking rhizome of water spinach with 5mL of hexane solution for 6h to remove cuticle on the plant surface, and washing the surface with PBS buffer solution for 3 times to remove hexane and cuticle residues; SDS was diluted to 5wt% with PBS buffer and perfused at the hollow site for 7 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.5wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted mixed solution of Triton-X-100 and sodium chlorite, wherein the mass percent of Triton-X-100 is 0.5wt% and the mass percent of sodium chlorite is 10wt%, flushing the rhizome hollow parts and the outside of plants by using the mixed solution of 10mLTriton-X-100 and sodium chlorite for 24 hours, exchanging the new mixed solution of Triton-X-100 and sodium chlorite every 12 hours, and flushing the surface 3 times by using the PBS buffer solution to remove Triton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 72h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: the mixture is stirred in a dark place by using PBS according to the concentration of 7 weight percent, the rotating speed of a magnetic stirrer is 500r/min, the temperature is 40 ℃ and the time is 15min, and a clear solution is obtained; human primary human VSMC (cells purchased from icellbiosciences inc, shanghai) were selected, expanded using 75T flasks using dedicated medium (purchased from icellbiosciences inc), and passaged using T75 flasks when cell density reached 90%; after the cell number reached 90%, pancreatin was treated to give a concentration of 10 5 cell/cm 2 After obtaining a cell pellet by using a centrifuge, discarding the supernatant; cell pellet was reconstituted to a concentration of 10 using Gelma solution 5 cell/cm 2 Is preserved in a dark place at 4 ℃ in a sterile condition andand finishing the use within 1 h. Before use, 0.3wt% of Irgacure is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in fig. 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 80r/min, a spray head is sprayed at a speed of 0.2 mu L/s, and continuous 365nm wave band light is matched for curing. Stopping after spraying for 1s, strengthening and curing for 5s, starting the spraying process again, repeating the process for 2 times, soaking the obtained Glema-VSMC decellularized artificial blood vessel in PBS, and preserving at 4 ℃.
FIG. 3 is a photograph of a Glema-VSMC decellularized vascular prosthesis prepared in this example taken by a cryo-electron microscope, with significant tissue venation and cell distribution.
Example 4
Taking root and stem of water spinach with the length of 3cm and the inner diameter of 2mm, soaking in ultrapure water at the temperature of 4 ℃ for 6 hours to remove surface stains; passing a glass rod with a length of 5cm and a diameter of 2mm through the hollow part as a support, diluting hexane to 90wt% with PBS buffer solution to obtain hexane solution, soaking rhizome of water spinach with 10mL of hexane solution for 12h to remove cuticle on the plant surface, and washing the surface with PBS buffer solution for 3 times to remove hexane and cuticle residues; SDS was diluted to 8wt% with PBS buffer and perfused at the hollow site for 5 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.1wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted solution of Triton-X-100 and sodium chlorite, flushing hollow parts of rhizomes and the outside of plants by using the mixed solution of 10mLTriton-X-100 and sodium chlorite with the mixed solution of Triton-X-100 and sodium chlorite of which the mass percentage is 0.5wt% and the mass percentage of sodium chlorite is 10wt% by using the mixed solution of Triton-X-100 and sodium chlorite for 48 hours, and then flushing the surface 3 times by using the PBS buffer solution every 12 hours to remove TTriton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 48h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: it was prepared at 10wt% using PBS% concentration is stirred in a dark place, the rotating speed of a magnetic stirrer is 1000r/min, the temperature is 40 ℃ and the time is 5min, and clear solution is obtained; human primary human VSMC (cells purchased from icellbiosciences inc, shanghai) were selected, expanded using 75T flasks using dedicated medium (purchased from icellbiosciences inc), and when cell density reached 90%, passaged using T75 flasks; after the cell number reached 90%, pancreatin was treated to give a concentration of 10 5 cell/cm 2 After obtaining a cell pellet by using a centrifuge, discarding the supernatant; cell pellet was reconstituted to a concentration of 10 using Gelma solution 6 cell/cm 2 Is stored aseptically at 4 ℃ in the dark and used within 1 hour. Before use, ru/SPS with the weight of 0.5 percent is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in the figure 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 120r/min, a spray head is sprayed at the speed of 0.3 mu L/s, and continuous light with the wave band of 550nm is matched for curing. Stopping after spraying for 1s, reinforcing and curing for 20s, starting the spraying process again, repeating the process for 3 times, soaking the obtained Glema-VSMC decellularized artificial blood vessel in PBS, and preserving at 4 ℃.
FIG. 5 is a stress-strain curve of GelMA used in the present example; FIG. 6 is a stress-strain curve of the decellularized scaffold prepared in this embodiment; FIG. 7 is a stress-strain curve of the Glema-VSMC decellularized vascular prosthesis prepared in this example; from fig. 5 to 7, it can be derived that: the Glema-VSMC acellular artificial blood vessel prepared by the embodiment has higher strength and meets the use standard of the artificial blood vessel.
Example 5
Taking root and stem of water spinach with the length of 5cm and the inner diameter of 3mm, soaking in ultrapure water at the temperature of 4 ℃ for 6 hours to remove surface stains; a glass bar with the length of 7cm and the diameter of 3mm is used as a support, hexane is diluted to 90wt% by using PBS buffer solution to obtain a hexane solution, 10mL of hexane solution is used for soaking the rhizome of the water spinach for 24 hours to remove cuticle on the surface of the plant, and the surface is washed 3 times by using the PBS buffer solution to remove hexane and cuticle residues; SDS was diluted to 10wt% with PBS buffer and perfused at the hollow site for 7 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.5wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted mixed solution of Triton-X-100 and sodium chlorite, wherein the mass percent of Triton-X-100 is 0.5wt% and the mass percent of sodium chlorite is 10wt%, flushing hollow parts of rhizomes and the outside of plants by using the mixed solution of 10mLTriton-X-100 and sodium chlorite for 48 hours, replacing the new mixed solution of Triton-X-100 and sodium chlorite every 12 hours, and flushing the surface 3 times by using PBS buffer solution to remove Triton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 72h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: the mixture is stirred in a dark place by using PBS according to the concentration of 5 weight percent, the rotating speed of a magnetic stirrer is 800r/min, the temperature is 40 ℃ and the time is 60min, and a clear solution is obtained; human primary human VSMC (cells purchased from icellbiosciences inc, shanghai) were selected, expanded using 75T flasks using dedicated medium (purchased from icellbiosciences inc), and when cell density reached 90%, passaged using T75 flasks; after the cell number reached 90%, pancreatin was treated to give a concentration of 10 6 cell/cm 2 After obtaining a cell pellet by using a centrifuge, discarding the supernatant; cell pellet was reconstituted to a concentration of 10 using Gelma solution 6 cell/cm 2 Is stored aseptically at 4 ℃ in the dark and used within 1 hour. Before use, ru/SPS with the weight percentage of 1 percent is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in the figure 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 200r/min, a spray head is sprayed at the speed of 0.5 mu L/s, and continuous light with the wave band of 550nm is matched for curing. Stopping after spraying for 2s, strengthening and curing for 10s, starting the spraying process again, repeating the process for 4 times, soaking the obtained Glema-VSMC decellularized artificial blood vessel in PBS, and preserving at 4 ℃.
FIG. 8 shows the DPAI photo-staining results of nuclei after 1 day co-culture of Glema-VSMC decellularized vascular prosthesis extract prepared in this example with endothelial cells.
Comparative example 1
Taking root and stem of water spinach with the length of 3cm and the inner diameter of 2mm, soaking in ultrapure water at the temperature of 4 ℃ for 6 hours to remove surface stains; passing a glass rod with a length of 5cm and a diameter of 2mm through the hollow part as a support, diluting hexane to 90wt% with PBS buffer solution to obtain hexane solution, soaking rhizome of water spinach with 10mL of hexane solution for 12h to remove cuticle on the plant surface, and washing the surface with PBS buffer solution for 3 times to remove hexane and cuticle residues; SDS was diluted to 8wt% with PBS buffer and perfused at the hollow site for 5 days, after which the surface was rinsed 3 times with PBS buffer to remove SDS and tissue residues; diluting Triton-X-100 to 0.1wt% by using PBS buffer solution, preparing a mixed solution of Triton-X-100 and sodium chlorite by using the diluted mixed solution of Triton-X-100 and sodium chlorite, wherein the mass percent of Triton-X-100 is 0.5wt% and the mass percent of sodium chlorite is 10wt%, flushing hollow parts of rhizomes and the outside of plants by using the mixed solution of 10mLTriton-X-100 and sodium chlorite for 48 hours, replacing the new mixed solution of Triton-X-100 and sodium chlorite every 12 hours, and flushing the surface 3 times by using PBS buffer solution to remove Triton-X-100, sodium chlorite and tissue residues; the decellularized scaffold was soaked with sterile deionized water for 48h. The resulting decellularized scaffolds were stored in sterile deionized water at 4 ℃.
Methacrylic acid hydrogel (Gelma) was selected as the surface modifying material: the mixture is stirred in a dark place by using PBS according to the concentration of 10 weight percent, the rotating speed of a magnetic stirrer is 1000r/min, the temperature is 40 ℃ and the time is 5min, and a clear solution is obtained; before use, ru/SPS with the weight of 0.5 percent is added in a dark place to serve as a photoinitiator, a hollow bar of the decellularized scaffold is inserted into a structural device shown in the figure 1, the hollow bar is fixed on a rotating shaft of a spraying device, the rotating speed is set to be 120r/min, a spray head is sprayed at the speed of 0.3 mu L/s, and continuous light with the wave band of 550nm is matched for curing. Stopping after spraying for 1s, strengthening and curing for 20s, starting the spraying process again, repeating the process for 3 times, soaking the obtained Glema-decellularized artificial blood vessel in PBS, and preserving at 4 ℃.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.

Claims (10)

1. The preparation method of the plant decellularized artificial blood vessel is characterized by comprising the following steps of:
the plant roots and/or stems with the hollow structure are decellularized to obtain a decellularized scaffold;
coating the photopolymerized hydrogel-exosome suspension on the outer surface of the acellular bracket under the condition of illumination to carry out a photocuring reaction so as to obtain the plant acellular artificial blood vessel; the wavelength of the illumination is 365-550 nm; the photopolymerized hydrogel-exosome suspension includes a photopolymerized hydrogel solution, an exosome, and a photoinitiator.
2. The method of claim 1, wherein the photopolymerizable hydrogel in the photopolymerizable hydrogel solution comprises one or more of methacrylic anhydride gelatin, methacryloylated hyaluronic acid, methacryloylated collagen, tyramine substituted hyaluronic acid, tyramine collagen, and tyramine gelatin.
3. The method of claim 1, wherein the exosome source comprises one or more of blood serum, bone marrow mesenchymal stem cells, adipose stem cells, vascular endothelial cell sources, and vascular contractile smooth muscle cell sources.
4. The method according to claim 1 or 2, wherein the mass percentage of the photopolymerizable hydrogel contained in the photopolymerizable hydrogel solution is 5 to 10wt%.
5. A method of preparation according to claim 1 or 3, wherein the mass concentration of the source of the exosome in the photopolymerised hydrogel-exosome suspension is from 2 to 200 μg/μl.
6. The method of claim 1, wherein the photoinitiator comprises one or more of Irgacure2959, VA-086, eosin Y, and Ru/SPS; in the photopolymerized hydrogel-exosome suspension, the mass percentage of the photoinitiator is 0.1-1 wt%.
7. The method of manufacturing according to claim 1, wherein the coating is a spray, and the operating parameters of the spray include: the rotation speed of the decellularized scaffold is 50-200 r/min; the speed of the photopolymerized hydrogel-exosome suspension sprayed out by the spray head is 0.1-2 mu L/s.
8. The method of claim 7, wherein the spraying is performed intermittently, each spraying having a duration of 1 to 3 seconds; the interval time between two adjacent spraying is 5-60 s.
9. The method of claim 1, wherein the decellularizing comprises the steps of:
using a glass rod to penetrate through a hollow part of a plant root and/or stem with a hollow structure as a support, and then immersing the plant root and/or stem with the hollow structure in a hexane solution to remove horny layers on the surface of the plant root and/or stem, thereby obtaining exfoliating roots and/or stems;
pouring sodium dodecyl sulfate solution into the hollow structure of the exfoliating root and/or stem to perform pretreatment for removing plant cell tissues, so as to obtain pretreated root and/or stem;
washing the outer surface of the pretreatment root and/or stem and the inner surface of the hollow structure by using a mixed solution of polyethylene glycol octyl phenyl ether and sodium chlorite, and carrying out treatment for removing plant cell tissues to obtain the decellularized scaffold.
10. The plant decellularized artificial blood vessel prepared by the preparation method of any one of claims 1 to 8, wherein the plant decellularized artificial blood vessel comprises a decellularized scaffold and a hydrogel-exosome photocuring coating attached to the decellularized scaffold.
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