CN115944786A - Integrated double-layer small vascular graft and preparation method thereof - Google Patents
Integrated double-layer small vascular graft and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an integrated double-layer small vascular graft, wherein the inner diameter of a pipe of the small vascular graft is 1-6 mm, the radial thickness of the pipe wall is 1-1.5 mm, the pipe wall from the inner wall of the pipe to the pipe wall with the radial thickness of 100-200 mu m is an inner layer of the pipe wall, an outer layer of the pipe wall is arranged between the outer wall of the pipe and the inner layer of the pipe wall, the inner layer of the pipe wall comprises electrostatic spinning submicron fibers, and bacterial cellulose nanofibers are interwoven in the inner pores of the electrostatic spinning submicron fibers to form the inner layer of the pipe wall with a nano/submicron fiber structure; the outer layer of the pipe wall is bacterial cellulose nanofibers containing micropores, the bacterial cellulose nanofibers on the inner layer and the outer layer of the pipe wall penetrate through each other to form a mutually-penetrated structure, and the two layers have binding force, so that the possibility of relative sliding and layering is avoided, and the stability of the structure of the graft can be maintained. The preparation process has the obvious advantages of simplicity, easy operation, low cost, less environmental pollution and the like.
Description
Technical Field
The invention belongs to the field of biological materials, and relates to an integrated double-layer small vascular graft and a preparation method thereof.
Background
Healthy blood vessels play an important role in maintaining normal physiological activities of the human body. According to the latest statistics of the world health organization, cardiovascular diseases have become one of the highest morbidity and mortality worldwide, and seriously threaten human health. The blood vessel transplantation operation is a commonly used treatment means in clinic at present. This type of surgery requires the use of the patient's own blood vessels (such as the great saphenous vein, the bilateral internal thoracic arteries, the radial artery, etc.), but the own blood vessels have the disadvantages of limited sources, limited access length, etc., and thus artificially synthesized vascular grafts have been developed. Although artificial vascular grafts (such as polyethylene terephthalate and expanded polytetrafluoroethylene) constructed by synthetic materials have been successfully applied to large-caliber (< 6 mm) blood vessels, intimal hyperplasia and thrombus are easily formed in small-caliber (< 6 mm) artificial blood vessels, and the long-term patency of the blood vessels is low, so that the preparation of small-caliber artificial vascular grafts with more excellent performance is not easy.
Studies have shown that constructing small vessel grafts that mimic the natural vascular structure is considered an effective solution to this problem. The natural blood vessel can be divided into three layers, each layer has different structure and function, and the layers are closely connected. Among them, the basal intima layer to which the endothelial cells of the monolayer adhere and the intermediate layer on which the vascular smooth muscle cells depend play an extremely important role in the function of the blood vessel. The applicant publicly reports the preparation of a biomimetic natural inner membrane from bacterial cellulose and electrospun fiber in a composite manner (reference 1.
To date, a series of methods have been proposed to prepare vascular grafts with excellent performance, but delamination between each layer is difficult to avoid during the manufacturing process of the multilayered structure of the artificial blood vessel (reference 2. When the effective connection between layers is lacked, not only the interlaminar stripping is easy to cause, the blood seeps out from the fiber clearance and pseudo aneurysm is caused, but also the transmission of interlaminar force is lacked, and the perception of endothelial cells to the morphological structure of the graft can be influenced, thereby influencing the antithrombotic capacity.
Disclosure of Invention
In view of the prior art, the invention provides an integrated double-layer small blood vessel graft, which comprises a tube wall inner layer and a tube wall outer layer, wherein the tube wall inner layer is composed of submicron fibers and bacterial cellulose nanofibers which are interwoven with each other, the tube wall outer layer is bacterial cellulose with micron-sized macropores, and the bacterial cellulose nanofibers penetrate through the two layers of the tube wall outer layer and the tube wall inner layer, so that interlayer binding force is provided to form an integrated structure. In the preparation process, a submicron fiber tube and a bacterial cellulose solution containing microspheres are co-cultured by a membrane-liquid interface culture method, the diameters of the submicron fibers and the microspheres are controlled, so that the bacterial cellulose nanofibers grow in the inner pores and the outer surfaces of the submicron fiber vascular graft, the microspheres only exist in the outer layer of the bacterial cellulose, the microspheres are removed in the purification and sterilization process in the later stage, an inner layer formed by interweaving the submicron fibers and the nanofibers and an outer layer formed by the bacterial cellulose with micron macropores are obtained, and the bacterial cellulose nanofibers penetrate through the inner layer and the outer layer to form the integrated double-layer small vascular graft. The integrated double-layer small vascular graft obtained by the invention has the remarkable advantages of simple operation, low cost, less environmental pollution and the like.
In the invention, the relationship between the bacterial cellulose and the bacterial cellulose nanofiber is as follows: the bacterial cellulose is a porous reticular nano-scale biopolymer polymer synthesized by microbial fermentation, and consists of unique filamentous fibers, and the fiber diameters of the unique filamentous fibers are in a nano scale, so the unique filamentous fibers are called as bacterial cellulose nanofibers.
In order to solve the technical problems, the invention provides an integrated double-layer small vascular graft, the inner diameter of a pipe is 1-6 mm, the radial thickness of the pipe wall is 1-1.5 mm, the pipe wall from the inner wall of the pipe to the pipe wall with the radial thickness of 100-200 mu m is an inner layer of the pipe wall, an outer layer of the pipe wall is arranged between the outer wall of the pipe and the inner layer of the pipe wall, the inner layer of the pipe wall comprises electrostatic spinning submicron fibers, and bacterial cellulose nanofibers are interwoven in inner pores of the electrostatic spinning submicron fibers to form the inner layer of the pipe wall with a nano/submicron fiber structure; the outer layer of the tube wall is bacterial cellulose nano-fibers containing micro-macropores, and the bacterial cellulose nano-fibers of the inner layer and the outer layer of the tube wall are mutually penetrated to provide interlayer binding force to form an integrated small vascular graft with a double-layer structure; according to the small blood vessel graft, a submicron fiber tube and bacterial cellulose bacteria liquid containing microspheres are co-cultured by a membrane liquid interface culture method, the diameters of the submicron fiber and the microspheres are controlled, so that the bacterial cellulose nanofibers grow in the inner pores and the outer pores of the submicron fiber, the microspheres only exist on the outer layer of the tube wall, and then the microspheres are removed while the bacteria are purified and sterilized, so that the inner layer of the tube wall formed by interweaving the submicron fiber and the bacterial cellulose nanofibers and the outer layer of the tube wall containing the bacterial cellulose nanofibers with micron macropores are obtained, and the bacterial cellulose nanofibers penetrate through the inner layer and the outer layer of the tube wall.
Further, the integrated double-layer small vascular graft provided by the invention is characterized in that the diameter of the electrostatic spinning submicron fiber is 500-900 nm; the diameter of the micron macropore is 50-300 μm.
Meanwhile, the invention also provides a method for preparing the integrated double-layer small vascular graft, which comprises the following process steps:
step one, preparing microspheres with the diameter of 50-300 microns, wherein the microspheres are one of gelatin microspheres, starch microspheres and paraffin microspheres, and sterilizing the microspheres for later use;
step two, preparing a submicron fiber tube by electrostatic spinning: preparing a submicron fiber tube with the inner diameter of 1-6 mm, the wall thickness of 100-200 mu m and the fiber diameter of 500-900 nm by adopting an electrostatic spinning process; drying the submicron fiber tube in an oven at 60 ℃ for 1-2 days, and sterilizing for later use;
step three, preparing an oxygen permeation sleeve mold: preparing an oxygen permeation tube A with the same outer diameter as the inner diameter of the submicron fiber tube prepared in the step two, preparing an oxygen permeation tube B with the inner diameter 2-3 mm larger than the outer diameter of the oxygen permeation tube A, coaxially sleeving the oxygen permeation tube B outside the oxygen permeation tube A to form an oxygen permeation sleeve, fixing the bottom of the oxygen permeation sleeve in a culture dish, wherein the upper end of the oxygen permeation sleeve is a feed inlet, and the feed inlet is provided with a plug;
step four, sterilizing the ultraviolet lamp of the oxygen permeation casing mould for 20 minutes, dripping 50-150 mu L of culture medium containing bacterial cellulose liquid into the oxygen permeation casing from the feed inlet, screwing the plug, vertically putting the oxygen permeation casing into a biochemical culture box, and culturing for 2 hours at 30 ℃, wherein the bottom of the oxygen permeation casing obtains a bacterial cellulose base membrane; sleeving the submicron fiber pipe prepared in the step two outside the oxygen permeation pipe A, wherein the bottom end of the submicron fiber pipe is in contact with the surface of the bacterial cellulose base membrane at the bottom of the oxygen permeation pipe A; under the aseptic condition, dripping a microsphere-containing bacterial cellulose culture solution with the mass volume ratio of 0.01-0.4 g/mL into a gap between the outer surface of a submicron fiber tube and an oxygen-permeable tube B from a feed inlet of the oxygen-permeable tube B by adopting a membrane-liquid interface culture method every 2-4 hours according to the amount of 50-150 microliter, wherein the bacterial cellulose culture solution is simultaneously diffused into the submicron fiber tube, and bacterial cellulose nanofibers simultaneously grow in the submicron fiber tube and the gap between the submicron fiber tube and the oxygen-permeable tube B until the bacterial cellulose nanofibers grow to the top of the oxygen-permeable tube;
and step five, taking the product prepared in the step four out of the oxygen permeation sleeve mold, and removing the microspheres while purifying and sterilizing to obtain the double-layer small blood vessel graft with the inner layer of the tube wall of the nano/submicron fiber structure and the outer layer of the tube wall of the bacterial cellulose nanofiber containing micron macropores, wherein the bacterial cellulose nanofiber penetrates through the inner layer and the outer layer to form the integral double-layer small blood vessel graft.
Further, the method for preparing the integrated double-layer small vessel graft comprises the following steps:
in the first step, the gelatin microspheres are prepared according to the following steps: adding gelatin into deionized water according to the mass volume ratio of 0.3g/mL, and stirring and dissolving in a water bath at 50 ℃ to form a gelatin solution; adding sorbitan monooleate into liquid paraffin according to the mass volume ratio of 0.02g/mL, and stirring in a water bath at 50 ℃ for 10 minutes to prepare a mixed solution A; adding the gelatin solution into the mixed solution A according to a volume ratio of 3; in the ice-water bath environment, adding formaldehyde into the mixed solution B according to a volume ratio of 2; taking out the mixture from the ice water bath, stirring at room temperature for 1 hour, moving to a fume hood, and standing for 24 hours to allow the mixture to be layered; removing the upper layer liquid, cleaning the lower layer product with absolute ethyl alcohol for 3 times until no turbidity is formed and layering, sequentially cleaning the filtered product with deionized water, isopropanol and deionized water for 3 times respectively, and freeze-drying to obtain gelatin microspheres; and (3) screening the gelatin microspheres with the size of 50-300 microns by using a stainless steel sieve.
In the second step, the sub-micron fiber tube is made of any one of Cellulose Acetate (CA), polyether sulfone (PES) and polyether ether ketone (PEEK).
In the third step, the oxygen permeation tube A and the oxygen permeation tube B are any one of an oxygen permeation silicone tube, a polydimethylsiloxane tube and a bulked polytetrafluoroethylene tube.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method has the advantages of simple preparation process, easy operation and low cost. The bacterial cellulose nanofiber in the integrated double-layer small vascular graft penetrates through the stent to form a mutually-penetrated structure, and binding force exists between the two layers, so that the possibility of relative sliding and layering can be avoided, and the stability of the graft structure can be maintained.
Drawings
FIG. 1 is a schematic structural view of an oxygen permeable casing mold in example 1 of the present invention;
FIG. 2 is a schematic structural view of the integrated double-layer small vessel graft of the present invention;
FIG. 3 is an SEM photograph of the inner and outer layers penetrated by the bacterial cellulose nanofibers prepared in example 1 of the present invention;
fig. 4 is an SEM photograph of the micro-macroporous bacterial cellulose prepared in example 2 of the present invention.
Detailed Description
The design idea of the integrated double-layer small vascular graft provided by the invention is as follows: the inner layer of the graft is composed of submicron fibers and bacterial cellulose nanofibers which are interwoven with each other, the outer layer is bacterial cellulose with micron-sized macropores, and the bacterial cellulose nanofibers penetrate through the inner layer and the outer layer to provide interlayer binding force so as to form an integrated structure. In order to prepare the integrated double-layer small blood vessel graft, the invention co-cultures a submicron fiber tube and bacterial cellulose bacteria liquid containing microspheres by a membrane-liquid interface culture method, and the diameters of the submicron fiber and the microspheres are controlled, so that the bacterial cellulose nano fibers only exist in the outer layer of the bacterial cellulose while growing in the inner pores and the outer layer of the submicron fiber blood vessel graft, and the microspheres are removed during purification and sterilization at the later stage, thereby obtaining an inner layer in which the submicron fiber and the nano fiber are mutually interwoven and an outer layer of the bacterial cellulose with micron macropores, and the bacterial cellulose nano fiber penetrates through the inner layer and the outer layer to form the integrated double-layer small blood vessel graft. The integrated double-layer small vascular graft obtained by the invention has the obvious advantages of simple operation, low cost, less environmental pollution and the like, and the double-layer small vascular graft can promote the growth of endothelial cells and the growth of smooth muscle cells by using the micro-macropore as an artificial blood vessel. In addition, because the inner layer and the outer layer of the tube wall are mutually interwoven by the nano fibers to form effective connection between layers, blood cannot seep out of fiber gaps and pseudo aneurysm caused by interlayer stripping, the transmission of interlayer force is enhanced, the perception of endothelial cells to the morphological structure of the graft is ensured, and the antithrombotic capacity is improved.
Based on the thought, the pipe inner diameter of the integrated small vascular graft provided by the invention is 1-6 mm, the radial thickness of the pipe wall is 1-1.5 mm, the pipe wall from the pipe inner wall to the pipe wall with the radial thickness of 100-200 mu m is a pipe wall inner layer, a pipe wall outer layer is arranged between the pipe outer wall and the pipe wall inner layer, the pipe wall inner layer comprises electrostatic spinning submicron fibers with the diameter of 500-900 nm, and the inner pores of the electrostatic spinning submicron fibers are interwoven with bacterial cellulose nanofibers to form the pipe wall inner layer with a nano/submicron fiber structure; the outer layer of the tube wall is bacterial cellulose nanofiber containing micro macropores with the diameter of 50-300 mu m, and the bacterial cellulose nanofiber on the inner layer and the outer layer of the tube wall are mutually penetrated to provide interlayer binding force to form an integrated small vascular graft with a double-layer structure; according to the small blood vessel graft, a submicron fiber tube and bacterial cellulose bacteria liquid containing microspheres are co-cultured by a membrane liquid interface culture method, the diameters of the submicron fiber and the microspheres are controlled, so that the bacterial cellulose nanofibers grow in the inner pores and the outer pores of the submicron fiber, the microspheres only exist on the outer layer of the tube wall, and then the microspheres are removed while the bacteria are purified and sterilized, so that the inner layer of the tube wall formed by interweaving the submicron fiber and the bacterial cellulose nanofibers and the outer layer of the tube wall of the bacterial cellulose nanofibers containing micron macropores are obtained, and the bacterial cellulose nanofibers penetrate through the inner layer and the outer layer of the tube wall, as shown in fig. 2.
In the invention, the microsphere is one of gelatin microsphere, starch microsphere and paraffin microsphere. The sub-micron fiber tube is made of any one of Cellulose Acetate (CA), polyether sulfone (PES) and polyether ether ketone (PEEK).
The invention will be further described with reference to the following figures and specific examples, which are not intended to limit the invention in any way.
Example 1, an integrated double-layered small vessel graft was prepared by the following process steps:
step one, preparing gelatin microspheres with the diameter of 100-250 microns: adding 4.5g of gelatin into 15mL of deionized water, and stirring and dissolving in a water bath at 50 ℃ to form a gelatin solution; 1g of sorbitan monooleate was added to 50mL of liquid paraffin, and stirred in a water bath at 50 ℃ for 10 minutes. Adding the prepared gelatin solution into a mixed solution of liquid paraffin and sorbitan monooleate, stirring in a water bath at 50 ℃ for 6 minutes, taking out the mixed solution, sealing with a preservative film, stirring in an ice water bath for 8 minutes, adding 2mL of formaldehyde into the mixed solution, stirring for 10 minutes, and then dropwise adding 20wt% of NaOH to adjust the pH value to 9 to obtain a mixture. The mixture was taken out from the ice-water bath, stirred at room temperature for 1 hour, moved to a fume hood, and left to stand for 24 hours, to allow the mixture to separate into layers. And after removing the upper layer liquid, cleaning the lower layer product with absolute ethyl alcohol for 3 times until no turbidity is formed and layering, sequentially cleaning the filtered product with deionized water, isopropanol and deionized water for 3 times respectively, and freeze-drying to obtain the gelatin microsphere. Sieving out gelatin microsphere particles with the size of 100-250 microns by using a stainless steel sieve, and sterilizing for later use.
Step two, preparing a cellulose acetate submicron fiber tube by electrostatic spinning: 5mL of acetic acid, 5mL of acetone, and 2.1g of cellulose acetate were taken, and the mixture was put into a vial, and stirred with a stirrer by magnetic force until completely dissolved. The ambient temperature is controlled at 35 ℃ and the humidity is controlled below 40%. A roller with the diameter of 4mm is used as a receiver, the spinning solution is injected into a 10mL injector, the distance between the emitter and the receiver is adjusted to be 20cm, the advancing speed of the solution is 4mL/h, the rotating speed of the roller is 100rpm, the positive pressure is adjusted to be 10kV, and the negative pressure is adjusted to be 3.7kV. The cellulose acetate submicron fiber tube with the spun thickness of 200 mu m (namely the inner diameter is 4mm, the outer diameter is 4.4 mm) and the fiber diameter of 500-900 nm is taken down from the roller, and is put into a drying oven at 60 ℃ for drying for 2 days, and the cellulose acetate submicron fiber tube is used for standby after sterilization treatment.
Step three, preparing an oxygen permeable silica gel casing mould: taking an oxygen-permeable silicone tube A with the outer diameter of 4mm, the thickness of 0.5mm (namely, the inner diameter of 3 mm) and the length of 80mm and an oxygen-permeable silicone tube B with the outer diameter of 7mm, the thickness of 0.5mm (namely, the inner diameter of 6 mm) and the length of 60mm, and vertically placing the oxygen-permeable silicone tube B in the oxygen-permeable silicone tube B to form a coaxial oxygen-permeable silicone sleeve. The bottom parts of the oxygen-permeable silicone tube A and the oxygen-permeable silicone tube B are fixed in a culture dish, a gap of 1mm is formed between the oxygen-permeable silicone tube B and the oxygen-permeable silicone tube A, a movable plug is arranged at the upper ends of the oxygen-permeable silicone tube A and the oxygen-permeable silicone tube B to serve as a feed inlet, so that an oxygen-permeable silicone sleeve mold shown in figure 1 is formed, and in figure 1, the meanings of reference signs are as follows: 1 is a movable plug, 2 is an oxygen permeable silicone tube B,3 is an oxygen permeable silicone tube A,4 is a gap between the oxygen permeable silicone tube B and the oxygen permeable silicone tube A, and 5 is a culture dish.
Step four, preparing a bacterial cellulose culture medium: 25g of glucose, 7.5g of yeast powder, 10g of peptone and 10g of sodium dihydrogen phosphate are sequentially added into a beaker filled with 1000mL of ultrapure water, and stirred until the glucose, the peptone and the sodium dihydrogen phosphate are completely dissolved. Then, a certain amount of glacial acetic acid is dripped into the beaker, and the pH is adjusted to 4-5. Placing the prepared culture medium into a sterilization pot, and sterilizing at 115 deg.C and 0.1MPa for 30 min.
Step five, preparing a bacterial cellulose base membrane: and (5) placing the oxygen permeable silica gel casing mould prepared in the step three into a super clean bench for ultraviolet lamp sterilization for 20 minutes. And (3) dripping 50 mu L of bacterial cellulose bacteria liquid raw material and 50 mu L of culture medium obtained from the step four into the gap between the oxygen permeable silicone tube B and the oxygen permeable silicone tube A of the oxygen permeable silicone sleeve, screwing a plug, vertically putting the oxygen permeable silicone sleeve into a biochemical incubator, and culturing for 2 hours at 30 ℃, thus obtaining the bacterial cellulose base membrane at the bottom of the silicone tube.
And step six, sleeving the cellulose acetate sub-micron fiber tube prepared in the step two outside the oxygen permeable silica gel tube A of the oxygen permeable silica gel sleeve, wherein the bottom end of the cellulose acetate sub-micron fiber tube is contacted with the surface of the bacterial cellulose base membrane.
And seventhly, under an aseptic condition, dripping a bacterial cellulose culture solution containing gelatin microspheres (the content of the gelatin microspheres is 0.15 g/mL) into a gap between a cellulose acetate submicron fiber tube with the outer diameter of 4.4mm and an oxygen-permeable silica gel tube B with the inner diameter of 6mm from a feed inlet at the upper end of the oxygen-permeable silica gel sleeve by adopting a membrane-liquid interface culture method every 3 hours according to the amount of 100 microlitres, wherein the diameter of the gelatin microspheres is 100-250 microlitres larger than the pore between the submicron fibers in the cellulose acetate submicron fiber tube, so that the gelatin microspheres are only distributed in the gap between the outer surface of the cellulose acetate submicron fiber tube and the oxygen-permeable silica gel tube B, the culture medium can be diffused into the cellulose acetate submicron fiber tube, and the bacterial cellulose grows in the submicron fiber tube and the gap between the outer surface of the submicron fiber tube and the outer silica gel tube simultaneously until the bacterial cellulose grows to the top of the silica gel tube.
Step eight, purifying and sterilizing, and simultaneously removing microspheres: taking out the product prepared in the seventh step, namely the double-layer small vascular graft with the inner diameter of 4mm from the oxygen permeable silica gel sleeve mold, and then purifying, namely cleaning with deionized water and NaOH, soaking with tert-butyl alcohol, and carrying out vacuum freeze drying; simultaneously removing bacteria and gelatin microspheres to obtain a pipe wall with an inner layer of a nano/submicron fiber structure and an outer layer of a pipe wall of bacterial cellulose containing micron macropores, wherein the bacterial cellulose nanofiber penetrates through the inner layer and the outer layer to form an integrated double-layer small blood vessel graft, as shown in fig. 2. FIG. 3 is an SEM photograph of the bacterial cellulose nanofibers prepared in example 1 penetrating through the inner and outer layers.
The pipe inner diameter of the integrated small vascular graft finally prepared in the embodiment 1 is 4mm, the radial thickness of the pipe wall is 1.5mm, the pipe wall from the pipe inner wall to the pipe wall with the radial thickness of 200 mu m is the pipe wall inner layer, the pipe wall outer layer is arranged between the pipe outer wall and the pipe wall inner layer, the pipe wall inner layer comprises electrostatic spinning cellulose acetate submicron fibers with the diameter of 500-900 nm, and the inner pores of the cellulose acetate submicron fibers are interwoven with bacterial cellulose nano fibers to form the pipe wall inner layer with a nano/submicron fiber structure; the outer layer of the pipe wall is bacterial cellulose nanofiber containing micron macropores with the diameter of 100-250 mu m, the bacterial cellulose nanofiber on the inner layer and the outer layer of the pipe wall are mutually penetrated, and the schematic microstructure diagram of the cross section of the pipe wall is shown in a partial enlarged view on the right side in fig. 2.
In the preparation method of the invention, the oxygen permeable silicone tube in the oxygen permeable silicone sleeve can be changed into other materials, such as a polydimethylsiloxane tube or an expanded polytetrafluoroethylene tube.
Example 2, an integrated double-layered small vessel graft was prepared by the following process steps:
step one, preparing gelatin microspheres with the diameter of 50-150 microns, wherein the preparation process is different from that of the embodiment 1, and finally, sieving out gelatin microsphere particles with the size of 50-150 microns by using a stainless steel sieve, and sterilizing the gelatin microsphere particles for later use.
Step two, preparing the polyethersulfone submicron fiber tube through electrostatic spinning: 10mL of N, N-dimethylformamide and 3.0g of polyether sulfone are taken and put into a small bottle, a stirrer is placed in the small bottle, and the mixture is magnetically stirred until the mixture is completely dissolved. The environmental temperature is controlled to be 20-40 ℃, and the humidity is controlled to be below 50%. A roller with the diameter of 2mm is used as a receiver, the spinning solution is injected into a 10mL injector, the distance between the emitter and the receiver is adjusted to be 15cm, the advancing speed of the solution is 2.5mL/h, the rotating speed of the roller is 500rpm, the positive pressure is adjusted to be 15kV, and the negative pressure is adjusted to be 3kV. The polyethersulfone submicron fiber tube with the thickness of 100 mu m (namely the inner diameter is 2mm, the outer diameter is 2.2 mm) and the fiber diameter of 500-900 nm is taken down from the roller, put into a drying oven at 60 ℃ for drying for 2 days, and sterilized for later use.
Step three, preparing an oxygen permeable silicone casing mould, which is different from the embodiment 1 in that the outer diameter of the oxygen permeable silicone tube A is 2mm, the thickness is 0.5mm (namely the inner diameter is 1 mm), and the length is 60mm; the oxygen permeable silicone tube B has an outer diameter of 6mm, a thickness of 0.5mm (i.e. an inner diameter of 5 mm) and a length of 50mm; thereby forming a gap of 1.5mm between the oxygen permeable silicone tube B and the oxygen permeable silicone tube A.
Step four, preparing a bacterial cellulose culture medium, which is the same as the preparation process of the embodiment 1.
Step five, preparing a bacterial cellulose base membrane: the difference from the example 1 is that the bacterial cellulose base membrane is obtained at the bottom of the oxygen permeable silica gel casing by changing the bacterial cellulose bacterial fluid raw material and the culture medium dropped into the gap between the oxygen permeable silica gel tube B and the oxygen permeable silica gel tube A of the oxygen permeable silica gel casing into 30 muL of bacterial cellulose bacterial fluid raw material and 30 muL of culture medium.
And step six, sleeving the polyether sulfone submicron fiber tube prepared in the step two outside an oxygen permeable silica gel tube A of the oxygen permeable silica gel sleeve, wherein the bottom end of the polyether sulfone submicron fiber tube is contacted with the surface of the bacterial cellulose base membrane.
And seventhly, under an aseptic condition, dripping a bacterial cellulose culture solution containing gelatin microspheres (the content of the gelatin microspheres is 0.10 g/mL) into a gap between a polyether sulfone submicron fiber tube with the outer diameter of 2.2mm and an oxygen-permeable silica gel tube B with the inner diameter of 5mm from a feed inlet at the upper end of the oxygen-permeable silica gel sleeve by adopting a membrane-liquid interface culture method every 2 hours according to the amount of 50 microlitres, wherein the gelatin microspheres are only distributed in the gap between the outer surface of the polyether sulfone submicron fiber tube and the oxygen-permeable silica gel tube B, the culture medium can be diffused into the polyether sulfone submicron fiber tube, and the bacterial cellulose grows in the gap between the polyether sulfone submicron fiber tube and the outer surface of the submicron fiber tube and the outer silica gel tube simultaneously until the bacterial cellulose grows to the top of the tube.
And step eight, purifying and sterilizing, and simultaneously removing the microspheres, wherein the steps are the same as the steps in the example 1, so that the integrated double-layer small blood vessel graft is finally formed, and an SEM photograph of the micro-macroporous bacterial cellulose containing microspheres removed in the example is shown in FIG. 4.
The pipe inner diameter of the integrated small vascular graft finally prepared in the embodiment 2 is 2mm, the radial thickness of the pipe wall is 1.5mm, the pipe wall from the pipe inner wall to the pipe wall with the radial thickness of 100 microns is a pipe wall inner layer, a pipe wall outer layer is arranged between the pipe outer wall and the pipe wall inner layer, the pipe wall inner layer comprises electrostatic spinning polyether sulfone submicron fibers with the diameter of 500-900 nm, and bacterial cellulose nano fibers are interwoven in inner pores of the polyether sulfone submicron fibers to form the pipe wall inner layer with a nano/submicron fiber structure; the outer layer of the pipe wall is bacterial cellulose nanofiber containing micro macropores with the diameter of 50-150 mu m, and the bacterial cellulose nanofiber on the inner layer and the outer layer of the pipe wall are mutually penetrated.
Example 3 preparation of an integrated double-layer small vessel graft, the process steps were as follows:
step one, screening the purchased starch microspheres with a stainless steel sieve to obtain the starch microspheres with the size of 50-150 mu m, and sterilizing for later use.
Step two, preparing the polyethersulfone submicron fiber tube by electrostatic spinning: 10mL of N, N-dimethylformamide and 3.0g of polyethersulfone are taken and put into a small bottle, a stirrer is put in the small bottle, and the small bottle is magnetically stirred until the N, N-dimethylformamide and the polyethersulfone are completely dissolved. The ambient temperature is controlled at 40 ℃ and the humidity is controlled below 50%. A roller with the diameter of 3mm is used as a receiver, the spinning solution is injected into a 10mL injector, the distance between the emitter and the receiver is adjusted to be 15cm, the advancing speed of the solution is 2.5mL/h, the rotating speed of the roller is 500rpm, the positive pressure is adjusted to be 15kV, and the negative pressure is adjusted to be 3kV. Taking the polyethersulfone submicron fiber tube with the thickness of 100 micrometers (namely the inner diameter is 3mm, the outer diameter is 3.2 mm) and the fiber diameter of 500-900 nm from the roller, putting the tube into a drying oven at 60 ℃ for drying for 2 days, and sterilizing for later use.
Step three, preparing a polydimethylsiloxane sleeve mold, wherein the outer diameter of the polydimethylsiloxane tube A is 3mm, the thickness of the polydimethylsiloxane tube A is 0.5mm (namely the inner diameter of the polydimethylsiloxane tube A is 2 mm), and the length of the polydimethylsiloxane tube A is 60mm; the polydimethylsiloxane tube B had an outer diameter of 6mm, a thickness of 0.5mm (i.e., an inner diameter of 5 mm), and a length of 50mm; a polydimethylsiloxane tube A with the outer diameter of 3mm is vertically placed in a polydimethylsiloxane tube B with the inner diameter of 5mm, the bottoms of the two polydimethylsiloxane tubes are fixed in a culture dish, a gap of 1mm is formed between the two tubes, and a movable plug is arranged at the upper end of the two polydimethylsiloxane tubes and serves as a feed inlet, so that the polydimethylsiloxane sleeve mold is formed.
Step four, preparing a bacterial cellulose culture medium, which is the same as the preparation process of the embodiment 1.
Step five, preparing a bacterial cellulose base membrane: the difference from example 1 is that bacterial cellulose base membrane was finally obtained on the bottom of the polydimethylsiloxane sleeve by dropping bacterial cellulose inoculum raw material and 40. Mu.L of culture medium into the gap between the two tubes of the polydimethylsiloxane sleeve mold, wherein the culture medium was 40. Mu.L of bacterial cellulose inoculum raw material and 40. Mu.L of culture medium.
And step six, sleeving the polyether sulfone submicron fiber pipe prepared in the step two outside the polydimethylsiloxane pipe A with the outer diameter of 3mm of the polydimethylsiloxane sleeve, wherein the bottom end of the pipe is contacted with the surface of the bacterial cellulose base membrane.
And seventhly, under an aseptic condition, dripping a bacterial cellulose culture solution containing starch microspheres (the content of the starch microspheres in the culture solution is 0.02 g/mL) into a gap between a polyether sulfone submicron fiber tube with the outer diameter of 3.2mm and a polydimethylsiloxane tube B with the inner diameter of 5mm from a feed inlet at the upper end of the mold every 2 hours by adopting a membrane-liquid interface culture method according to the amount of 80 microlitres, wherein the diameter of the starch microspheres is larger than the pore space between the submicron fibers in the submicron fiber tube, so that the starch microspheres are only distributed in the gap between the outer surface of the submicron fiber tube and the outer polydimethylsiloxane tube, the culture medium can be diffused into the submicron fiber tube, and the bacterial cellulose grows in the gaps between the submicron fiber tube, the outer surface of the submicron fiber tube and the outer polydimethylsiloxane tube at the same time until the bacterial cellulose grows to the top of the mold.
And step eight, purifying and sterilizing, and simultaneously removing the microspheres, which is basically the same as the step of the example 1, and finally forming the integrated double-layer small vascular graft.
The pipe inner diameter of the integrated small vascular graft finally prepared in the embodiment 3 is 3mm, the radial thickness of the pipe wall is 1mm, the pipe wall from the pipe inner wall to the pipe wall with the radial thickness of 100 microns is a pipe wall inner layer, a pipe wall outer layer is arranged between the pipe outer wall and the pipe wall inner layer, the pipe wall inner layer comprises electrostatic spinning polyether sulfone submicron fibers with the diameter of 500-900 nm, and bacterial cellulose nanofibers are interwoven in the inner pores of the polyether sulfone submicron fibers to form a pipe wall inner layer with a nano/submicron fiber structure; the outer layer of the pipe wall is bacterial cellulose nanofiber containing micro macropores with the diameter of 50-150 mu m, and the bacterial cellulose nanofiber on the inner layer and the outer layer of the pipe wall are mutually penetrated.
Although the present invention is described in detail with reference to the drawings, the present invention is not limited to the above-mentioned embodiments, the integrated double-layered small vascular graft with inner diameter of 2mm,3mm and 4mm prepared in the above-mentioned examples is only illustrative and not restrictive, and the person skilled in the art can prepare the integrated double-layered small vascular graft with inner diameter of 1-6 mm by changing the radial dimension of two oxygen permeable tubes in the oxygen permeable casing mold according to the preparation method of the present invention. Further, it is possible to make changes without departing from the gist of the present invention, and these are within the scope of the present invention.
Claims (6)
1. An integrated double-layer small blood vessel graft is characterized in that the inner diameter of a tube of the small blood vessel graft is 1-6 mm, the radial thickness of the tube wall is 1-1.5 mm, the tube wall from the inner wall of the tube to the tube wall with the radial thickness of 100-200 mu m is an inner layer of the tube wall, an outer layer of the tube wall is arranged between the outer wall of the tube and the inner layer of the tube wall, the inner layer of the tube wall comprises electrostatic spinning submicron fibers, and bacterial cellulose nanofibers are interwoven in inner pores of the electrostatic spinning submicron fibers to form the inner layer of the tube wall with a nano/submicron fiber structure; the outer layer of the tube wall is bacterial cellulose nano-fibers containing micro-macropores, and the bacterial cellulose nano-fibers of the inner layer and the outer layer of the tube wall are mutually penetrated to provide interlayer binding force to form an integrated small vascular graft with a double-layer structure;
according to the small blood vessel graft, a submicron fiber tube and bacterial cellulose bacteria liquid containing microspheres are co-cultured through a membrane liquid interface culture method, the diameters of the submicron fiber and the microspheres are controlled, so that the bacterial cellulose nanofibers grow in the inner pores and the outer pores of the submicron fiber, the microspheres only exist in the outer layer of the tube wall, and then the microspheres are removed through purification and sterilization, so that the inner layer of the tube wall formed by interweaving the submicron fiber and the bacterial cellulose nanofibers and the outer layer of the tube wall containing the bacterial cellulose nanofibers with micron macropores are obtained, and the bacterial cellulose nanofibers penetrate through the inner layer and the outer layer of the tube wall.
2. The integrated double-layer small vessel graft according to claim 1, wherein the electrospun sub-micron fibers have a diameter of 500-900 nm; the diameter of the micron macropore is 50-300 μm.
3. Method for preparing an integrated double-layered small vessel graft according to claim 1, characterized in that the following process steps are used:
step one, preparing a microsphere with the diameter of 50-300 mu m, wherein the microsphere is one of gelatin microsphere, starch microsphere and paraffin microsphere, and sterilizing for later use;
step two, preparing a submicron fiber tube by electrostatic spinning: preparing a submicron fiber tube with the inner diameter of 1-6 mm, the wall thickness of 100-200 mu m and the fiber diameter of 500-900 nm by adopting an electrostatic spinning process; drying the submicron fiber tube in an oven at 60 ℃ for 1-2 days, and sterilizing for later use;
step three, preparing an oxygen permeation sleeve mold: preparing an oxygen permeation tube A with the same outer diameter as the inner diameter of the submicron fiber tube prepared in the second step, preparing an oxygen permeation tube B with the inner diameter 2-3 mm larger than the outer diameter of the oxygen permeation tube A, coaxially sleeving the oxygen permeation tube B outside the oxygen permeation tube A to form an oxygen permeation sleeve, fixing the bottom of the oxygen permeation sleeve in a culture dish, wherein the upper end of the oxygen permeation sleeve is a feed port, and the feed port is provided with a plug;
step four, sterilizing the ultraviolet lamp of the oxygen permeation casing mould for 20 minutes, dripping 50-150 mu L of culture medium containing bacterial cellulose liquid into the oxygen permeation casing from the feed inlet, screwing the plug, vertically putting the oxygen permeation casing into a biochemical culture box, and culturing for 2 hours at 30 ℃, wherein the bottom of the oxygen permeation casing obtains a bacterial cellulose base membrane; sleeving the submicron fiber pipe prepared in the step two outside the oxygen permeation pipe A, wherein the bottom end of the submicron fiber pipe is in contact with the surface of the bacterial cellulose base membrane at the bottom of the oxygen permeation pipe A;
under the aseptic condition, dripping a microsphere-containing bacterial cellulose culture solution with the mass volume ratio of 0.01-0.4 g/mL into a gap between the outer surface of a submicron fiber tube and an oxygen-permeable tube B from a feed inlet of the oxygen-permeable tube B by adopting a membrane-liquid interface culture method every 2-4 hours according to the amount of 50-150 microliter, wherein the bacterial cellulose culture solution is simultaneously diffused into the submicron fiber tube, and bacterial cellulose nanofibers simultaneously grow in the submicron fiber tube and the gap between the submicron fiber tube and the oxygen-permeable tube B until the bacterial cellulose nanofibers grow to the top of the oxygen-permeable tube;
and step five, taking the product prepared in the step four out of the oxygen permeation sleeve mold, and removing the microspheres while purifying and sterilizing to obtain the double-layer small blood vessel graft with the inner layer of the tube wall of the nano/submicron fiber structure and the outer layer of the tube wall of the bacterial cellulose nanofiber containing micron macropores, wherein the bacterial cellulose nanofiber penetrates through the inner layer and the outer layer to form the integral double-layer small blood vessel graft.
4. The method for preparing an integrated double-layer small vessel graft according to claim 3, wherein in the first step, the gelatin microspheres are prepared according to the following steps:
adding gelatin into deionized water according to the mass-to-volume ratio of 0.3g/mL, and stirring and dissolving in a water bath at 50 ℃ to form a gelatin solution; adding sorbitan monooleate into liquid paraffin according to the mass volume ratio of 0.02g/mL, and stirring in a water bath at 50 ℃ for 10 minutes to prepare a mixed solution A;
adding the gelatin solution into the mixed solution A according to a volume ratio of 3; in the ice-water bath environment, adding formaldehyde into the mixed solution B according to a volume ratio of 2;
taking out the mixture from the ice water bath, stirring at room temperature for 1 hour, moving to a fume hood, and standing for 24 hours to allow the mixture to be layered; removing the upper layer liquid, cleaning the lower layer product with absolute ethyl alcohol for 3 times until no turbidity and layering exist, sequentially cleaning the filtered product with deionized water, isopropanol and deionized water for 3 times respectively, and freeze-drying to obtain gelatin microspheres; and (3) screening the gelatin microspheres with the size of 50-300 microns by using a stainless steel sieve.
5. The method for preparing an integrated double-layer small vessel graft according to claim 3, wherein in the second step, the submicron fiber tube is made of any one of Cellulose Acetate (CA), polyether sulfone (PES) and polyether ether ketone (PEEK).
6. The method for preparing the integral double-layer small vessel graft according to claim 3, wherein in the third step, the oxygen permeation tube A and the oxygen permeation tube B are any one of an oxygen permeation silicone tube, a polydimethylsiloxane tube and a bulked polytetrafluoroethylene tube.
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