CN115122690A - Preparation method of nerve conduit - Google Patents
Preparation method of nerve conduit Download PDFInfo
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- CN115122690A CN115122690A CN202210754118.8A CN202210754118A CN115122690A CN 115122690 A CN115122690 A CN 115122690A CN 202210754118 A CN202210754118 A CN 202210754118A CN 115122690 A CN115122690 A CN 115122690A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
- B29D—PRODUCING PARTICULAR ARTICLES FROM PLASTICS OR FROM SUBSTANCES IN A PLASTIC STATE
- B29D23/00—Producing tubular articles
- B29D23/001—Pipes; Pipe joints
Abstract
The invention relates to the technical field of nerve conduits, in particular to a preparation method of a nerve conduit. The preparation method comprises the following steps: filling paraffin wax microspheres into the tubular space of the mould; the mold comprises a shell with a columnar inner wall and a column, wherein the column is arranged in the shell, and the tubular space is formed between the column and the shell; heating the paraffin wax microspheres to obtain a paraffin wax network; wrapping extracellular matrix outside the paraffin network to obtain an outer layer structure; injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture; and carrying out freeze-drying treatment on the solid-liquid mixture, removing the paraffin network, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with the double-layer composite structure. The embodiment of the invention provides a preparation method of a nerve conduit, and provides the nerve conduit capable of improving the nerve repair effect and accelerating the nerve repair efficiency.
Description
Technical Field
The invention relates to the technical field of nerve conduits, in particular to a preparation method of a nerve conduit.
Background
Peripheral nerve defects are one of the most common clinical trauma, and this type of nerve injury can cause complete dual loss of sensory and motor functions in the distal limb innervated by the affected nerve, resulting in severe disability, which can have a profound impact on both the quality of work and life of the patient.
In the related technology, for peripheral nerve defects with short distance clinically, on the basis of the principle of tension-free suture, the method of directly suturing the broken ends of nerves is mostly adopted for repairing; for long-distance peripheral nerve defects, autologous nerve transplantation is often adopted for repair. However, autologous nerve transplantation can achieve only partial recovery of nerve function, and the repair efficiency is low.
Therefore, in order to overcome the above disadvantages, a method for preparing a nerve conduit is urgently needed.
Disclosure of Invention
The embodiment of the invention provides a preparation method of a nerve conduit, and provides the nerve conduit capable of improving the nerve repair effect and accelerating the nerve repair efficiency.
The embodiment of the invention provides a preparation method of a nerve conduit, which comprises the following steps:
filling paraffin wax microspheres into the tubular space of the mould; the mold comprises a shell and a column body, wherein the inner wall of the shell is columnar, the column body is arranged in the shell, and the tubular space is formed between the column body and the shell;
heating the paraffin wax microspheres to obtain a paraffin wax network;
wrapping extracellular matrix outside the paraffin network to obtain an outer layer structure;
injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture;
and performing freeze-drying treatment on the solid-liquid mixture, removing the paraffin network, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with the double-layer composite structure.
In one possible design, before the filling the paraffin wax microspheres into the tubular space of the mold, the method further comprises:
immersing the mold in a polyethylene oxide solution;
and drying the mould immersed in the polyethylene oxide solution.
In one possible design, the immersing the mold in a polyethylene oxide solution includes:
immersing a shell included in the mold into a polyethylene oxide solution with the concentration of 15-30 wt%;
immersing a cylinder included in the mold into a polyethylene oxide solution with the concentration of 8-12 wt%;
wherein the solvent of the polyethylene oxide solution is ethanol.
In a possible design, the temperature of the heating treatment is 40-60 ℃, and the time of the heating treatment is 40-100 min.
In one possible design, after the obtaining the paraffin network, before the wrapping the extracellular matrix outside the paraffin network, the method further includes:
immersing the mold containing the paraffin network in absolute ethanol;
removing the wax network from the mold;
cleaning and drying the mould;
and (3) immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt%, and taking out.
In one possible design, the biological protein solution is a silk fibroin solution with a concentration of 10-15 wt%.
In one possible design, the bioprotein solution is a collagen gel at a concentration of 0.8 to 1.4 wt%.
In one possible design, the lyophilizing the solid-liquid mixture and removing the paraffin network includes:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
soaking the freeze-dried tube body in 90 wt% methanol for 2 hours;
and (3) cleaning the paraffin wax network in the freeze-drying pipe body by using n-hexane.
In one possible design, the lyophilizing the solid-liquid mixture and removing the paraffin network includes:
performing freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
performing cross-linking treatment on the freeze-drying tube body;
and cleaning the paraffin wax network in the freeze-drying pipe body by using normal hexane.
In one possible design, after the obtaining of the nerve conduit, the method further includes:
injecting a 2-10 wt% egg yolk lecithin solution into the inner layer structure;
and carrying out vacuum drying treatment on the nerve conduit injected with the egg yolk lecithin solution.
Compared with the prior art, the invention at least has the following beneficial effects:
in this embodiment, paraffin microspheres are filled into a tubular space formed by a shell and a cylinder, so that the paraffin microspheres form a tubular body in the tubular space, and then the paraffin microspheres are subjected to heating treatment, so that the surfaces of the paraffin microspheres are melted and adhered to each other, thereby forming a paraffin network; the extracellular matrix is wrapped outside the paraffin network to obtain an outer layer structure, and comprises the basement membrane, so that the extracellular matrix can provide conditions for the immigration of Schwann cells with the basement membrane, the Schwann cells can secrete neurotrophic factors to promote the survival of damaged neurons and the regeneration of axons of the damaged neurons, and participate in the formation of nerve fibers in a peripheral nervous system. Injecting a biological protein solution into the space of the paraffin network wrapped by the outer layer structure, filling the biological protein solution into the reticular gaps of the paraffin network to obtain a solid-liquid mixture, carrying out freeze-drying treatment on the solid-liquid mixture, changing the biological protein solution filled in the paraffin network into a reticular solid, removing the paraffin network to obtain the nerve conduit with the outer layer structure being extracellular matrix and the inner layer structure being a reticular structure prepared from biological protein, wherein the reticular structure prepared from biological protein is degradable, and the degradation rate is matched with the tissue regeneration rate; the freeze-drying treatment enables the net-shaped structure to have good supporting performance, can play a good supporting role on external extracellular matrix, and solves the problems of collapse, poor regeneration, scar tissue formation, hyperplasia, adhesion and the like caused by the ischemia of the extracellular matrix. The inner layer reticular structure has a porous structure and high porosity, is beneficial to the permeation of bioactive factors and provides nutrition for the growth of capillary vessels and fibrous tissues; the biological protein has good biocompatibility, and can enable cells to be quickly adhered and proliferated by matching with a net structure so as to improve the efficiency of nerve repair.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a flow chart of a method for manufacturing a nerve conduit according to an embodiment of the present invention;
FIG. 2 is a flow chart of another method for preparing a nerve conduit according to an embodiment of the present invention;
FIG. 3 is a flow chart of a method for preparing a nerve conduit according to another embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a mold according to an embodiment of the present invention;
FIG. 5 is an electron microscope image of the inner mesh structure of the nerve conduit according to the embodiment of the present invention;
fig. 6 is an electron microscope image of an inner mesh structure of another nerve conduit according to an embodiment of the present invention.
In the figure:
1-a shell;
2-column body.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.
In the description of the embodiments of the present invention, unless explicitly specified or limited otherwise, the terms "first", "second", and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance; the term "plurality" means two or more unless specified or indicated otherwise; the terms "connected," "fixed," and the like are to be construed broadly and may, for example, be fixedly connected, detachably connected, integrally connected, or electrically connected; may be directly connected or indirectly connected through an intermediate. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the description of the present invention, it should be understood that the directional terms such as "upper" and "lower" used in the embodiments of the present invention are described with reference to the angles shown in the drawings, and should not be construed as limiting the embodiments of the present invention. In addition, in this context, it will also be understood that when an element is referred to as being "on" or "under" another element, it can be directly on "or" under "the other element or be indirectly on" or "under" the other element via an intermediate element.
As shown in fig. 1, an embodiment of the present invention provides a method for preparing a nerve conduit, including:
106, injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture;
and 108, performing freeze-drying treatment on the solid-liquid mixture and removing the paraffin network to obtain an inner layer structure inside the outer layer structure so as to obtain the nerve conduit with the double-layer composite structure.
In the embodiment, paraffin wax microspheres are filled into a tubular space formed by a shell 1 and a cylinder 2 to form a tubular body in the tubular space, and then the paraffin wax microspheres are subjected to heating treatment to melt the surfaces of the paraffin wax microspheres and further adhere to each other to form a paraffin wax network; the extracellular matrix is wrapped outside the paraffin network to obtain an outer layer structure, and comprises the basement membrane, so that the extracellular matrix can provide conditions for the immigration of Schwann cells with the basement membrane, the Schwann cells can secrete neurotrophic factors to promote the survival of damaged neurons and the regeneration of axons of the damaged neurons, and participate in the formation of nerve fibers in a peripheral nervous system. Injecting a biological protein solution into a paraffin network space wrapped by the outer layer structure, filling the biological protein solution into net-shaped gaps of the paraffin network to obtain a solid-liquid mixture, performing freeze-drying treatment on the solid-liquid mixture, changing the biological protein solution filled in the paraffin network into net-shaped solid, removing the paraffin network to obtain a nerve conduit with an extracellular matrix as an outer layer structure and a net-shaped structure (figures 5 and 6) prepared from biological protein as an inner layer structure, wherein the net-shaped structure prepared from the biological protein is degradable, and the degradation rate is matched with the tissue regeneration rate; the freeze-drying treatment enables the net structure to have good supporting performance, can play a good role in supporting external extracellular matrix, and solves the problems of collapse, poor regeneration, scar tissue formation, hyperplasia, adhesion and the like caused by ischemia of the extracellular matrix. The inner layer reticular structure has a porous structure and high porosity, is beneficial to the permeation of bioactive factors and provides nutrition for the growth of capillary vessels and fibrous tissues; the biological protein has good biocompatibility, and can enable cells to be quickly adhered and proliferated by matching with a net structure so as to improve the efficiency of nerve repair.
The porosity of the reticular structure prepared by the method is 70-85%, and the specific surface area is 15-21 m 2 /g。
In this example, the preparation method of the paraffin wax microsphere is as follows:
adding paraffin blocks into 0.5% (mass to volume) polyvinyl alcohol solution, wherein 100ml of polyvinyl alcohol solution is added into every 5g of paraffin blocks, heating and stirring at the constant temperature of 60-90 ℃ at 60-380 r/min after adding the paraffin blocks, forming paraffin microspheres uniformly dispersed in the solution after the paraffin is completely melted, stopping heating, adding ice blocks, continuously stirring for a plurality of minutes, sieving by using a gradient sieve 35 meshes (the diameter of the sieve is 500 micrometers), 50 meshes (300 micrometers), 70 meshes (212 micrometers) and 140 meshes (106 micrometers), washing away the polyvinyl alcohol by using running water, dehydrating and drying by using alcohol to prepare the paraffin microspheres with different particle sizes, and storing at 4 ℃ for later use after subpackaging.
The stirring speed and the concentration of the polyvinyl alcohol solution in the manufacturing process can influence the grain diameter of the paraffin globule, and in a certain range, the faster the stirring speed and the higher the polyvinyl alcohol concentration are, the smaller the grain diameter of the prepared paraffin globule is, and the more the shape of the paraffin globule tends to be spherical; on the contrary, the slower the stirring speed, the lower the polyvinyl alcohol concentration, the larger the particle size of the produced paraffin wax pellet, and the higher the proportion of non-spherical shapes. The optimal stirring speed after adding the paraffin blocks is 180r/min, the optimal concentration of the polyvinyl alcohol solution is 0.5 percent (mass-to-volume ratio), the optimal heating temperature in the stirring process is 75 ℃, the paraffin prepared under the parameters is regular in microspherical shape, the yield of 200-300 micrometers is the highest, and the pore size of 100-300 micrometers is most beneficial to crawling substitution of cells.
In this embodiment, the material for preparing the extracellular matrix includes at least one of descending aorta, porcine heart, and porcine small intestine submucosa.
The method for preparing extracellular matrix by descending aorta is as follows:
(1) descending aorta of fresh slaughtered pork pig
Transporting the slaughtered animal to a laboratory for treatment at low temperature within 24 hours, specifically, removing connective tissues and fat on the periphery of the descending aorta by using a micro-scissors, ligating small branches by using a 9-0 suture line, taking out a blood vessel, washing the blood vessel by using heparinized normal saline for three times, placing the blood vessel in normal saline solution containing 1% polystyrene, and storing the blood vessel at the temperature of 4 ℃;
(2) cell processing
Cutting the blood vessel into 2-5 cm segments in an ultra-clean bench, placing the segments into a 0.075% sodium dodecyl sulfate solution, shaking the segments gently for 12 hours in a shaking table at room temperature, and washing the segments with physiological saline for 3 times, wherein each time lasts for 4 hours, so as to obtain the extracellular matrix.
The method for preparing extracellular matrix by porcine heart or porcine small intestine submucosa comprises the following steps:
(1) pretreatment of
Selecting non-invasive and uniform-thickness fresh heart anterior wall part or small intestine submucosa of pig, removing fat tissue, and coating pig heart or pigSoaking small intestine submucosa in deionized water, repeatedly rinsing for 3 times (5 min each time), removing residual adipose tissue with scissors, and trimming to about 10 × 10cm 2 Sizing to obtain a pretreated material;
(2) degreasing
Adjusting the temperature of a magnetic heating stirrer to 40 ℃, adding the magnetic heating stirrer into 0.4% of sodium dodecyl sulfate according to the material-liquid ratio of 2.5-5 (preferably 3), rotating at the rotating speed of 120r/min for 1.5h, and then washing twice according to the material-liquid ratio of 3-6 (preferably 4);
(3) alkali treatment
Adjusting the temperature of a magnetic heating stirrer to be 25-35 ℃, adding 0.2-1.4% (preferably, the concentration is 1.1%) NaOH solution according to the material-liquid ratio of 2.5-5 (preferably, 3), adjusting the rotating speed to be 120r/min, rotating for 30min, then rotating for 10min every 50min, rotating for 16h totally, and washing for three times according to the material-liquid ratio of 3-6 (preferably, 4) after treatment; it should be noted that the alkaline solution has a corrosion-removing effect, can destroy cytoplasmic components, is beneficial to removing part of immune components, and can cause serious damage to the fiber structure due to overhigh temperature.
(4) Dealkalization: adjusting the temperature of a magnetic heating stirrer to 30 ℃, adding 3.0% ammonium chloride solution according to the material-liquid ratio of 2.5-5 (preferably 3), adjusting the rotating speed to rotate for 1.5h, measuring the pH value every 30min until the pH value is neutral, and washing for three times according to the material-liquid ratio of 2.5-5 (preferably 3) after the treatment;
(5) enzyme softening: adjusting the temperature of a magnetic heating stirrer to 40 ℃, adding a pancreatin solution or a pepsin solution with the concentration of 0.2-0.5% according to the feed-liquid ratio of 2-3 (preferably 2.5), adjusting the rotating speed to rotate for 30min, adding a 1398 neutral protease solution or a Triton-X-100 solution with the concentration of 0.2-0.8% after the completion of the rotation for 1-4 h, and washing for 3 times according to the feed-liquid ratio of 2.5-5 (preferably 3) after the completion of the rotation;
(6) washing with PBS: adjusting the magnetic heating stirrer to room temperature (25 ℃), and cleaning for 3 times (15 min each time) according to the liquid ratio of 2.5-5 (preferably 3) to obtain extracellular matrix;
(7) and (3) sample preservation: the pericardium after decellularization was preserved in PBS buffer and placed in a refrigerator at 4 ℃.
In some embodiments of the present invention, before filling the paraffin wax microspheres into the tubular space of the mold, the method further comprises:
immersing the mold in a polyethylene oxide solution;
and drying the mould immersed in the polyethylene oxide solution.
In this embodiment, dip the mould in the polyethylene oxide solution, make the mould surface be covered with the polyethylene oxide solution, after carrying out drying process to the mould, the surface parcel one deck polyethylene oxide solid of mould, this solid has certain thickness, can occupy certain space, this space is headspace, so set up, after paraffin microballon forms paraffin network, wash the polyethylene oxide solid on mould surface after taking out paraffin microballon, can provide the space for the extracellular matrix of parcel at paraffin network outer wall, and then make the paraffin network of parcel extracellular matrix can pack into the tubular space of mould.
The thickness of the headspace can be adjusted by the concentration of the polyethylene oxide solution, and the higher the concentration of the solution, the thicker the thickness.
In some embodiments of the invention, dipping the mold into a polyethylene oxide solution comprises:
immersing a shell 1 included in the mold into a polyethylene oxide solution with the concentration of 15-30 wt%;
immersing a cylinder 2 included in the mold into a polyethylene oxide solution with the concentration of 8-12 wt%;
wherein, the solvent of the polyethylene oxide solution is ethanol.
In the embodiment, the shell 1 is immersed in a polyethylene oxide solution with the concentration of 15-30 wt% to obtain a reserved space with the thickness same as that of the extracellular matrix; the column body 2 is immersed in a polyethylene oxide solution with the concentration of 8-12 wt%, thin-layer polyethylene oxide wrapped outside the column body 2 can be obtained, and the thin-layer polyethylene oxide is only used for taking out the paraffin network conveniently in the later period, so that the thickness is thin, and the paraffin network is convenient to demould after being cleaned.
It will be appreciated that the concentration of the polyethylene oxide solution can be flexibly adjusted as required to accommodate different thicknesses of extracellular matrix.
The ethanol is selected as the solvent, so that the time for removing the solvent can be shortened, the preparation time of the nerve conduit is further shortened, and in addition, the ethanol is nontoxic after volatilization, so that the environment is not polluted and the human health is not harmed.
In some embodiments of the present invention, the temperature of the heating treatment is 40 to 60 ℃, and the time of the heating treatment is 40 to 100 min.
In the embodiment, the temperature of the heating treatment is close to or equal to the melting point of the paraffin, and the time of the heating treatment is 40-100 min at the heating temperature, so that the surface of the paraffin microspheres is melted, and the spherical shape can be maintained, and thus, adjacent paraffin microspheres are adhered to each other to form a paraffin network. If the temperature of the heat treatment is higher than 60 ℃ and/or the time of the heat treatment is longer than 100min, the paraffin microspheres cannot keep spherical, and if the temperature of the heat treatment is lower than 40 ℃ and/or the time of the heat treatment is shorter than 40min, the paraffin microspheres cannot melt and cannot be adhered to each other to form a paraffin network.
In some embodiments of the present invention, after obtaining the paraffin network, before wrapping the extracellular matrix outside the paraffin network, the method further comprises:
immersing a mold containing a paraffin network in absolute ethanol;
taking the paraffin wax network out of the mould;
cleaning and drying the mold;
and (3) immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt%, and taking out.
In this embodiment, the mold containing the wax network is immersed in absolute ethanol to facilitate removal of the wax network from the mold, and if the mold is coated with polyethylene oxide, the ethanol can also dissolve the coated polyethylene oxide to further facilitate removal of the wax network. The ethanol can also dissolve polyethylene oxide on the inner wall of the shell 1, can provide space for the extracellular matrix wrapped outside the paraffin network subsequently, and is convenient for loading the paraffin network wrapped with the extracellular matrix into a mold. And (3) immersing the dried mould into 8-12 wt% polyethylene oxide solution again, and taking out, so that when demoulding is needed in the subsequent preparation of the nerve conduit, the mould can be smoothly demoulded after being immersed in ethanol.
In some embodiments of the invention, the biological protein solution is a silk fibroin solution with a concentration of 10-15 wt%.
In this embodiment, silk fibroin has excellent biocompatibility, the degradation rate is matched with the tissue regeneration rate, and the higher the concentration of the silk fibroin solution is, the higher the density of the obtained mesh solid after freeze-drying is. If the concentration of the silk fibroin solution is higher than 15 wt%, the obtained reticulated solid has a low specific surface area, and if the concentration of the silk fibroin solution is lower than 10 wt%, the obtained reticulated solid has poor supportability.
The silk fibroin is a high-purity protein secreted by endothelial cells of the silk gland at the rear part of the silkworm, does not contain organelles and other biological impurities, and has high biological safety. Silk fibroin is composed of twenty amino acids such as alanine, serine and the like, has good biocompatibility, can be biodegraded, and finally, the degradation product is polypeptide and free amino acid which are easy to be metabolized by organisms. Studies show that the silk fibroin material can well support the growth of neurons and neural stem cells. The catheter material prepared from the silk fibroin has low inflammatory reaction in vivo and is degradable, and can promote the functional recovery of peripheral nerve defects.
In some embodiments of the invention, a method of preparing silk fibroin comprises:
adding mulberry silk into 0.02mol/L Na at 100 deg.C 2 CO 3 Boiling the solution twice, each time for 30min, washing with tap water and distilled water for several times to completely remove sericin, and air drying for later use. Dissolving degummed silk in 9.3mol/L LiBr solution, dissolving for 4h at 60 ℃, dialyzing for 3d (the molecular weight cutoff is 12000-14000) to obtain a fibroin solution with the concentration of about 8%, centrifuging for 15min at 12000rpm, concentrating a low-concentration PEG solution for a certain time to obtain a high-concentration fibroin solution, centrifuging for 15min at 12000rpm, centrifuging and measuring the concentration by a dry constant weight method. Storing in a refrigerator at 4 deg.C.
In some embodiments of the invention, the bioprotein solution is a collagen gel at a concentration of 0.8 to 1.4 wt%.
In this example, collagen has excellent biocompatibility, the degradation rate matches with the tissue regeneration rate, and the higher the concentration of collagen gel, the higher the density of the mesh solid obtained after lyophilization. If the concentration of the collagen gel is more than 1.4 wt%, the obtained reticular solid has a low specific surface area, and if the concentration of the collagen gel is less than 10 wt%, the obtained reticular solid has poor supportability.
In some embodiments of the present invention, the method of preparing the collagen gel comprises:
(1) removing redundant fascia, fat, muscle and the like on the bovine achilles tendon, washing with tap water, neatly arranging in a freezing box, freezing at-20 ℃ for at least 12 h;
(2) cutting frozen bovine achilles tendon into slices of about 1mm, placing in a filter screen, and washing until the liquid is clear;
(3) enzymolysis: carrying out enzymolysis on the cleaned bovine achilles tendon slices, and fully stirring for not less than 72 h; wherein the mass ratio of the enzymolysis liquid to the bovine achilles tendon is 130: 1, the volume ratio of purified water to acetic acid in the enzymolysis liquid is 25: 1, the mass ratio of the purified water to the pepsin is 15: 1.
(4) salting out: centrifuging the solution after enzymolysis, taking supernatant, adding the supernatant into sodium chloride solution to separate out white flocculent collagen, filtering and washing, and draining off water.
(5) And (3) dialysis: filling the salted-out material into a dialysis bag, wherein the volume of the salted-out material is about 1/3 of the dialysis bag; placing the dialysis bag in a dialysate of 0.057mol/L acetic acid solution for 6 days, surreptitiously heating to 10-20 deg.C, and replacing the dialysate every 3 days; then placing the dialysis bag in 0.00057mol/L acetic acid solution for dialysis for 5 days, wherein the dialysis temperature is 10-20 ℃, and the dialysate is replaced every 1 day; dialyzing in 0.0000057mol/L acetic acid solution at pH of 5.0-5.5 from day 12 at 10-20 deg.C, and changing the dialysate once a day as required.
(6) The collagen gel was removed and tested for solids content.
In some embodiments of the invention, lyophilizing the solid-liquid mixture and removing the paraffin network comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
soaking the freeze-dried tube body in methanol with the concentration of 90 wt% for 2 hours;
and (3) washing off the paraffin wax network in the freeze-dried pipe body by using n-hexane.
In this example, soaking the lyophilized tube in 90 wt% methanol for 2 hours can shape silk fibroin, increasing the support of the prepared mesh solid. In addition, the method for cleaning off the paraffin network can be a Soxhlet extraction method, and specifically, the paraffin network in the stent is cleaned off by using n-hexane, then the n-hexane is replaced by cyclohexane, and then the cyclohexane is removed by vacuumizing.
In some embodiments of the present invention, the lyophilization process comprises a prefreezing stage, a first sublimation stage, a second sublimation stage, and a cooling stage, wherein the process conditions of each stage are as follows:
a pre-freezing stage: the target temperature is-12 to-8 ℃, the speed is 3 to 4.0 ℃/min, and the constant temperature duration is 280 to 320 min;
a first sublimation stage: vacuumizing, wherein the air is mixed at 90-110 Pa, the target temperature is-4 to-2 ℃, the speed is 0.6-0.8 ℃/min, and the constant temperature duration is 1300-1340 min;
the second sublimation stage, the evacuation, 90 ~ 110Pa aerify, including five intensification ladders, do respectively:
the temperature is between-1 and 1 ℃, the speed is 0.2 to 0.3 ℃/min, and the constant temperature duration is 110 to 130 min;
the temperature is 8-12 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;
the temperature is 18-22 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;
the temperature is 28-32 ℃, the speed is 1.0-1.2 ℃/min, and the constant temperature duration is 110-130 min;
38-42 ℃, the speed is 1.0-1.2 ℃/min, the constant temperature duration is as follows: judging the end point every 10 minutes until the end point is qualified; the end point is judged to be less than or equal to 0.9Pa/10 min;
and (3) cooling: cooling to room temperature at a rate of 1.4-1.6 deg.C/min.
In the embodiment, the pre-cooling stage can be carried out for 10-14 h at-20 ℃ and 2-6 h at-70 ℃ or for 3-10 min in liquid nitrogen, and the rest steps are carried out for freeze drying according to the process.
In some embodiments of the invention, lyophilizing the solid-liquid mixture and removing the paraffin network comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
performing cross-linking treatment on the freeze-drying pipe body;
and (4) cleaning the paraffin wax network in the freeze-dried pipe body by using normal hexane.
In this embodiment, carry out cross-linking treatment to freeze-drying body and can avoid collagen to meet the water and take place the swelling and destroy netted solid's structure, increase the stability of the netted solid who makes. In addition, the method for cleaning off the paraffin network can be a Soxhlet extraction method, and specifically, the paraffin network in the stent is cleaned off by using n-hexane, then the n-hexane is replaced by cyclohexane, and then the cyclohexane is removed by vacuumizing.
In some embodiments of the invention, the process of cross-linking treatment comprises:
a. preparing a cross-linking agent solution: measuring edible alcohol with a certain volume by using a measuring cylinder, transferring the edible alcohol into a reaction kettle, transferring a glutaraldehyde solution with a certain volume by using a liquid transfer gun (the mass fraction of glutaraldehyde is 50%), and mixing the glutaraldehyde solution with the liquid transfer gun according to the volume ratio of the edible alcohol to the glutaraldehyde solution of 100: preparing alcohol solution of glutaral at the ratio of 0.05;
b. soaking the materials in the crosslinking solution, wherein the stirring speed of the reaction kettle is 20-30r/min, and the uppermost layer of the materials can rotate with stirring, and crosslinking is carried out for 48 h. (note: after the material adsorbed the crosslinking agent and sunk during crosslinking, if the material was below the paddle, the paddle did not contact the material, the stirring speed was reduced to 10r/min.
c. Water washing step
(1) Taking out the cross-linked material from the cross-linking agent solution;
(2) the centrifuge was loaded with filter bags and centrifuged once (5 seconds); the rotating speed of the centrifugal machine is 3000r/min by default;
(3) soaking in 3% hydrogen peroxide solution at a ratio of 1:80 for 42 + -1 hr, wherein the volume (ml) of the 3% hydrogen peroxide solution is equal to the mass (g) multiplied by 80 of the product, and the product is completely soaked in the hydrogen peroxide solution.
(4) Carrying out ultrasonic treatment on purified water for 30min, and centrifuging once (5s) after ultrasonic treatment; repeating for 2 times;
(5) ultrasonic treating with edible alcohol for 5min, and immersing the product. After the ultrasonic treatment is finished, the centrifugal operation is carried out once (5 seconds);
d. vacuum drying
And (3) putting the centrifuged material into a vacuum drying oven for vacuum drying, wherein the vacuum degree is not higher than-0.08 Mpa, the temperature is set to be 50 ℃, and the drying is carried out for at least 24 hours.
In some embodiments of the present invention, after obtaining the nerve conduit, further comprising:
injecting a yolk lecithin solution with the concentration of 2-10 wt% into the inner layer structure;
the nerve conduit injected with the yolk lecithin solution is subjected to vacuum drying treatment.
In this embodiment, the yolk lecithin is digested to release choline, choline and acetyl coenzyme are catalyzed by choline acetyltransferase to generate acetylcholine, the acetylcholine is a chemical transmitter of all synapses between pre-ganglionic and postganglionic fibers of the autonomic nervous system, muscle nerve joints, all postganglionic fibers of parasympathetic ganglia and postganglionic fibers of sympathetic nerves, and the increase of the content of the acetylcholine can promote the rapid enhancement of cerebral nerve synapses, thereby accelerating the information transmission speed among nerve cells in the brain and further improving the memory and learning ability of a human. In addition, egg yolk lecithin can be used for treating multiple neurosis. If the weight part of the egg yolk lecithin is less than 2 wt%, the nerve repairing effect is poor, and if the weight part of the egg yolk lecithin is more than 10 wt%, the porosity of the network structure is reduced.
It should be noted that the egg yolk lecithin solution may be injected into the net structure by a syringe.
As shown in fig. 2, the present invention also provides another method for preparing a nerve conduit, comprising:
202, immersing a cylinder 2 included in the mold into a polyethylene oxide solution with the concentration of 8-12 wt%;
208, heating the paraffin microspheres to obtain a paraffin network;
step 226, washing off the paraffin network in the freeze-dried pipe body by using normal hexane to obtain an inner layer structure inside the outer layer structure so as to prepare the nerve conduit with the double-layer composite structure;
step 228, injecting a 2-10 wt% egg yolk lecithin solution into the inner layer structure;
and step 230, performing vacuum drying treatment on the nerve conduit injected with the yolk lecithin solution.
As shown in fig. 3, an embodiment of the present invention further provides a method for preparing a nerve conduit, including:
308, heating the paraffin microspheres to obtain a paraffin network;
step 322, performing freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
step 326, washing the paraffin network in the freeze-drying pipe body by using normal hexane, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with the double-layer composite structure;
328, injecting a yolk lecithin solution with the concentration of 2-10 wt% into the inner layer structure;
and step 330, carrying out vacuum drying treatment on the nerve conduit injected with the egg yolk lecithin solution.
In order to more clearly illustrate the technical solution and advantages of the present invention, a method for manufacturing a nerve conduit is described in detail below with reference to several embodiments.
Example 1
(1) Mold pretreatment
Respectively immersing a shell and a cylinder which are included by the mould into polyethylene oxide (molecular weight 2000) solution with the concentration of 20 wt% and 10 wt%, taking out and drying; wherein the solvent is ethanol;
(2) preparation of Paraffin wax network
Filling the paraffin microspheres into the mould treated in the step (1), slightly oscillating, and heating at 50 ℃ for 60min to obtain a paraffin network;
(3) encapsulating extracellular matrix
Immersing the mould in ethanol, gently taking out the paraffin wax network, and wrapping the extracellular matrix outside the paraffin wax network to obtain an outer layer structure;
(4) preparation of nerve conduit
Cleaning and drying the mould, placing the outer layer structure and the paraffin network wrapped by the outer layer structure in the mould, injecting a silk fibroin solution with the concentration of 13 wt%, performing freeze-drying treatment to obtain a freeze-dried body, soaking the freeze-dried body for 2 hours by using methanol with the concentration of 90%, and removing the paraffin network in the freeze-dried body to obtain the nerve conduit;
(5) compound egg yolk lecithin
Injecting 6 wt% of yolk lecithin solution into the inner layer structure to fill the yolk lecithin solution into the gaps of the reticular structure of the inner layer of the nerve conduit, and performing vacuum drying to obtain the nerve conduit of the composite yolk lecithin.
Example 2
Example 2 is essentially the same as example 1, except that:
in the step (1), a shell and a cylinder which are included in the mold are respectively immersed into a polyethylene oxide (molecular weight 2000) solution with a concentration of 15 wt% and a concentration of 10 wt%;
in the step (2), heat treatment is carried out for 40min at 40 ℃;
in the step (4), injecting silk fibroin solution with the concentration of 10 wt%;
in step (5), a 2 wt% egg yolk lecithin solution was injected into the inner layer structure.
Example 3
Example 3 is essentially the same as example 1, except that:
in the step (1), a shell and a cylinder which are included in the mold are respectively immersed into a 30 wt% concentration polyethylene oxide (molecular weight 2000) solution and a 12 wt% concentration polyethylene oxide solution;
in the step (2), heating treatment is carried out at 60 ℃ for 100 min;
in the step (4), injecting silk fibroin solution with the concentration of 15 wt%;
in step (5), a 10 wt% egg yolk lecithin solution was injected into the inner layer structure.
Example 4
Example 4 is essentially the same as example 1, except that:
and (4) cleaning and drying the mould, placing the outer layer structure and the paraffin network wrapped by the outer layer structure in the mould, injecting collagen gel with the concentration of 1.2 wt%, performing freeze-drying treatment to obtain a freeze-dried body, performing cross-linking treatment on the freeze-dried body, and removing the paraffin network in the freeze-dried body to obtain the nerve conduit.
Example 5
Example 5 is essentially the same as example 2, except that:
and (4) cleaning and drying the mould, placing the outer layer structure and the paraffin network wrapped by the outer layer structure into the mould, injecting collagen gel with the concentration of 0.8 wt%, performing freeze-drying treatment to obtain a freeze-dried body, performing cross-linking treatment on the freeze-dried body, and removing the paraffin network in the freeze-dried body to obtain the nerve conduit.
Example 6
Example 6 is essentially the same as example 3, except that:
in the step (4), the mould is cleaned and dried, the outer layer structure and the paraffin network wrapped by the outer layer structure are placed in the mould, collagen gel with the concentration of 1.4 wt% is injected, a freeze-dried body is obtained after freeze-drying treatment, cross-linking treatment is carried out on the freeze-dried body, and the nerve conduit is obtained after the paraffin network in the freeze-dried body is removed.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A method of preparing a nerve conduit, comprising:
filling paraffin wax microspheres into the tubular space of the mould; the mold comprises a shell and a column body, wherein the inner wall of the shell is columnar, the column body is arranged in the shell, and the tubular space is formed between the column body and the shell;
heating the paraffin wax microspheres to obtain a paraffin wax network;
wrapping extracellular matrix outside the paraffin network to obtain an outer layer structure;
injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture;
and carrying out freeze-drying treatment on the solid-liquid mixture, removing the paraffin network, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with the double-layer composite structure.
2. The method for preparing a paraffin wax microsphere according to claim 1, wherein before the filling of the paraffin wax microsphere into the tubular space of the mold, the method further comprises:
immersing the mold in a polyethylene oxide solution;
and drying the mould immersed in the polyethylene oxide solution.
3. The method of claim 2, wherein said immersing said mold in a polyethylene oxide solution comprises:
immersing a shell included in the mold into a polyethylene oxide solution with the concentration of 15-30 wt%;
immersing a cylinder included in the mold into a polyethylene oxide solution with the concentration of 8-12 wt%;
wherein the solvent of the polyethylene oxide solution is ethanol.
4. The method according to claim 1, wherein the temperature of the heat treatment is 40 to 60 ℃ and the time of the heat treatment is 40 to 100 min.
5. The method for preparing an extracellular matrix according to any one of claims 1 to 4, wherein after the obtaining of the paraffin network, before the wrapping of the extracellular matrix outside the paraffin network, the method further comprises:
immersing the mold containing the paraffin network in absolute ethanol;
removing the wax network from the mold;
cleaning and drying the mould;
and (3) immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt%, and taking out.
6. A method as claimed in any one of claims 1-4, wherein the biological protein solution is a silk fibroin solution with a concentration of 10-15 wt%.
7. The method for preparing a collagen gel according to any one of claims 1 to 4, wherein the bioprotein solution is a collagen gel having a concentration of 0.8 to 1.4 wt%.
8. The method for preparing according to claim 6, wherein the freeze-drying the solid-liquid mixture and removing the paraffin network comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
soaking the freeze-drying tube body in methanol with the concentration of 90 wt% for 2 hours;
and (3) cleaning the paraffin wax network in the freeze-drying pipe body by using n-hexane.
9. The method for preparing according to claim 7, wherein the freeze-drying the solid-liquid mixture and removing the paraffin network comprises:
performing freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
performing cross-linking treatment on the freeze-drying tube body;
and (3) cleaning the paraffin wax network in the freeze-drying pipe body by using n-hexane.
10. The method for preparing a nerve conduit according to any one of claims 1 to 4, further comprising, after the obtaining of the nerve conduit:
injecting a 2-10 wt% egg yolk lecithin solution into the inner layer structure;
and carrying out vacuum drying treatment on the nerve conduit injected with the egg yolk lecithin solution.
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| CN115122690B (en) | 2023-09-29 |
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