CN115122690B - Preparation method of nerve conduit - Google Patents
Preparation method of nerve conduit Download PDFInfo
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- CN115122690B CN115122690B CN202210754118.8A CN202210754118A CN115122690B CN 115122690 B CN115122690 B CN 115122690B CN 202210754118 A CN202210754118 A CN 202210754118A CN 115122690 B CN115122690 B CN 115122690B
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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
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Abstract
The invention relates to the technical field of nerve catheters, in particular to a preparation method of a nerve catheter. The preparation method comprises the following steps: filling paraffin microspheres into a tubular space of a mold; the die comprises a shell with a columnar inner wall and a column body, wherein the column body is arranged in the shell, and a tubular space is formed between the column body and the shell; heating the paraffin microspheres to obtain a paraffin network; wrapping an 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, which can provide the nerve conduit capable of improving the nerve repairing effect and accelerating the nerve repairing efficiency.
Description
Technical Field
The invention relates to the technical field of nerve catheters, in particular to a preparation method of a nerve catheter.
Background
Peripheral nerve defects are one of the most common wounds in clinic, and the nerve damage of the type can cause the far-end limb dominated by the affected nerve to have complete double loss of sensory and motor functions, thereby causing serious disability to occur and bringing great influence to the work and life quality of patients.
In the related art, for peripheral nerve defects with shorter distance clinically, on the basis of a tension-free suture principle, a nerve end-broken direct suture method is mostly adopted for repairing; in the case of long-distance peripheral nerve defects, an autologous nerve transplanting method is often adopted for repairing the peripheral nerve defects. However, the autologous nerve transplantation can only achieve restoration of a part of nerve functions, and repair efficiency is low.
Thus, in view of the above shortcomings, there is an urgent need for a method of preparing a nerve conduit.
Disclosure of Invention
The embodiment of the invention provides a preparation method of a nerve conduit, which can provide the nerve conduit capable of improving the nerve repairing effect and accelerating the nerve repairing efficiency.
The embodiment of the invention provides a preparation method of a nerve conduit, which comprises the following steps:
filling paraffin microspheres into a tubular space of a mold; the die comprises a shell with a columnar inner wall and a column body, wherein the column body is arranged in the shell, and a tubular space is formed between the column body and the shell;
heating the paraffin microspheres to obtain a paraffin network;
wrapping an 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.
In one possible design, before the filling of the paraffin microspheres into the tubular space of the mold, further comprises:
immersing the mold in a polyethylene oxide solution;
and drying the mold 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 die into a polyethylene oxide solution with a concentration of 15-30wt%;
immersing a column body included in the die into a polyethylene oxide solution with the concentration of 8-12 wt%;
wherein the solvent of the polyethylene oxide solution is ethanol.
In one possible design, the temperature of the heat treatment is 40-60 ℃ and the time of the heat treatment is 40-100 min.
In one possible design, after the obtaining of the paraffin network, before the encapsulating of the extracellular matrix outside the paraffin network, further comprising:
immersing the mold containing the paraffin network in absolute ethanol;
removing the paraffin network from the mould;
cleaning and drying the die;
immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt% and taking out.
In one possible design, the bioprotein solution is a silk fibroin solution having 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 comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
immersing the freeze-dried tube body in 90wt% methanol for 2 hours;
and cleaning the paraffin wax network in the freeze-dried tube body by using n-hexane.
In one possible design, the 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;
carrying out cross-linking treatment on the freeze-dried pipe body;
and cleaning the paraffin wax network in the freeze-dried tube body by using n-hexane.
In one possible design, after the obtaining the nerve conduit, the method further comprises:
injecting a yolk lecithin solution with a concentration of 2-10wt% into the inner layer structure;
and (3) carrying out vacuum drying treatment on the nerve conduit injected with the egg yolk lecithin solution.
Compared with the prior art, the invention has at least the following beneficial effects:
in the embodiment, the paraffin microspheres are filled into a tubular space formed by the shell and the column body, 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 then adhered to each other to form a paraffin network; the extracellular matrix is wrapped outside the paraffin network to obtain an outer layer structure, and the extracellular matrix comprises a basement membrane, so that the extracellular matrix can provide conditions for the migration of schwann cells also provided with the basement membrane, the schwann cells can secrete neurotrophic factors, the survival of damaged neurons and the regeneration of axons of the damaged neurons are promoted, the damaged neurons participate in the composition of nerve fibers in the peripheral nervous system, in addition, the extracellular matrix can also allow the exchange of moisture and nutrient substances, the non-nerve cells are prevented from entering the inside of the nerve conduit, and the extracellular matrix can promote the effect of nerve repair. Injecting a bioprotein solution into the paraffin network space wrapped by the outer layer structure, filling the bioprotein solution into network gaps of a paraffin network to obtain a solid-liquid mixture, performing freeze-drying treatment on the solid-liquid mixture, changing the bioprotein solution filled in the paraffin network into network solids, removing the paraffin network to obtain a nerve conduit with an outer layer structure being an extracellular matrix and an inner layer structure being a network structure prepared by bioprotein, wherein the network structure prepared by the bioprotein is degradable, and the degradation rate is matched with the tissue regeneration rate; the freeze-drying treatment ensures that the reticular structure has good supporting property, can play a good supporting role on the external extracellular matrix, and solves the problems of collapse, poor regeneration, formation of scar tissue, hyperplasia, adhesion and the like caused by ischemia of the extracellular matrix. The inner layer net structure has a porous structure and high porosity, is favorable for permeation of bioactive factors, and provides nutrition for capillary and fibrous tissue ingrowth; the biological protein has good biocompatibility, and can enable cells to be adhered and proliferated rapidly by matching with a reticular structure so as to improve the nerve repairing efficiency, and in addition, the nerve conduit also has high specific surface area and excellent surface physicochemical property, and can further improve the nerve repairing effect and efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of a method for preparing a nerve conduit according to an embodiment of the present invention;
FIG. 2 is a flowchart 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 of preparing a further nerve conduit according to an embodiment of the present invention;
FIG. 4 is a schematic structural view of a mold according to an embodiment of the present invention;
FIG. 5 is an electron microscope image of a mesh structure of an inner layer of a nerve conduit according to an embodiment of the present invention;
fig. 6 is an electron microscope image of another inner layer mesh structure of a nerve conduit according to an embodiment of the present invention.
In the figure:
1-a housing;
2-column.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.
In the description of embodiments of the present invention, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying any relative importance unless explicitly specified or limited otherwise; the term "plurality" means two or more, unless specified or indicated otherwise; the terms "connected," "secured," and the like are to be construed broadly, and may be, for example, fixedly connected, detachably connected, integrally connected, or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art according to the specific circumstances.
In the description of the present specification, it should be understood that the terms "upper", "lower", and the like used in the embodiments of the present invention are described in terms of the angles shown in the drawings, and should not be construed as limiting the embodiments of the present invention. In the context of this document, it will also be understood that when an element is referred to as being "on" or "under" another element, it can be directly on the other element or be indirectly on the other element through intervening elements.
As shown in fig. 1, an embodiment of the present invention provides a method for preparing a nerve conduit, including:
step 100, filling paraffin microspheres into a tubular space of a die; the die comprises a shell 1 with a columnar inner wall and a column body 2 (figure 4), wherein the column body 2 is arranged in the shell 1, and a tubular space is formed between the column body 2 and the shell 1;
step 102, heating the paraffin microspheres to obtain a paraffin network;
104, wrapping the extracellular matrix outside a paraffin network to obtain an outer layer structure;
step 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, 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.
In the embodiment, paraffin microspheres are filled into a tubular space formed by a shell 1 and a column 2, 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 then adhered to each other to form a paraffin network; the extracellular matrix is wrapped outside the paraffin network to obtain an outer layer structure, and the extracellular matrix comprises a basement membrane, so that the extracellular matrix can provide conditions for the migration of schwann cells also provided with the basement membrane, the schwann cells can secrete neurotrophic factors, the survival of damaged neurons and the regeneration of axons of the damaged neurons are promoted, the damaged neurons participate in the composition of nerve fibers in the peripheral nervous system, in addition, the extracellular matrix can also allow the exchange of moisture and nutrient substances, the non-nerve cells are prevented from entering the inside of the nerve conduit, and the extracellular matrix can promote the effect of nerve repair. Injecting a bioprotein solution into the paraffin network space wrapped by the outer layer structure, filling the bioprotein solution into network gaps of a paraffin network to obtain a solid-liquid mixture, performing freeze-drying treatment on the solid-liquid mixture, changing the bioprotein solution filled in the paraffin network into network solids, removing the paraffin network to obtain a nerve conduit with an outer layer structure being an extracellular matrix and an inner layer structure being a network structure (shown in fig. 5 and 6) prepared by bioprotein, wherein the network structure prepared by the bioprotein is degradable, and the degradation rate is matched with the tissue regeneration rate; the freeze-drying treatment ensures that the reticular structure has good supporting property, can play a good supporting role on the external extracellular matrix, and solves the problems of collapse, poor regeneration, formation of scar tissue, hyperplasia, adhesion and the like caused by ischemia of the extracellular matrix. The inner layer net structure has a porous structure and high porosity, is favorable for permeation of bioactive factors, and provides nutrition for capillary and fibrous tissue ingrowth; the biological protein has good biocompatibility, and can enable cells to be adhered and proliferated rapidly by matching with a reticular structure so as to improve the nerve repairing efficiency, and in addition, the nerve conduit also has high specific surface area and excellent surface physicochemical property, and can further improve the nerve repairing effect and efficiency.
The porosity of the network structure prepared by the method is 70-85%, and the specific surface area is 15-21 m 2 /g。
In this example, the paraffin microspheres were prepared as follows:
adding paraffin blocks into 0.5% (mass volume ratio) concentration polyvinyl alcohol solution, wherein 100ml polyvinyl alcohol solution is added into every 5g paraffin blocks, heating and stirring at constant temperature of 60-380 r/min after adding paraffin blocks, forming paraffin microspheres uniformly dispersed in the solution after paraffin is completely melted, stopping heating, adding ice cubes, continuing stirring for a plurality of minutes, sieving with a gradient sieve of 35 meshes (screen diameter of 500 mu m), 50 meshes (300 mu m), 70 meshes (212 mu m) and 140 meshes (106 mu m), washing polyvinyl alcohol with running water, dehydrating and airing with alcohol, preparing paraffin microspheres with different particle diameters, and storing at 4 ℃ for standby after split charging.
The stirring speed and the concentration of the polyvinyl alcohol solution in the manufacturing process can influence the particle size of the paraffin pellets, and in a certain range, the faster the stirring speed is, the higher the concentration of the polyvinyl alcohol is, the smaller the particle size of the prepared paraffin pellets is, and the shape of the paraffin pellets tends to be spherical; conversely, the slower the stirring rate, the lower the concentration of the polyvinyl alcohol, and the larger the particle size of the prepared paraffin pellets, the higher the proportion of non-spherical shapes. The optimal stirring speed after adding paraffin blocks is 180r/min, the optimal concentration of the polyvinyl alcohol solution is 0.5% (mass volume ratio), the optimal heating temperature in the stirring process is 75 ℃, the paraffin microspheres prepared under the parameters are regular in shape, the yield of 200-300 microns is highest, and the pore diameter of 100-300 microns is most favorable for crawling substitution of cells.
In this embodiment, the extracellular matrix preparation material includes at least one of a descending aorta, a pig heart, and a pig small intestine submucosa.
The method for preparing extracellular matrix by descending aorta is as follows:
(1) Fresh slaughtered pork pig descending aorta
Transporting the animals to a laboratory at a low temperature within 24 hours after slaughtering, specifically, stripping off connective tissues and fat at the periphery of descending aorta by micro-shearing, ligating tiny branches by using 9-0 suture lines, taking out blood vessels, flushing three times by using heparinized normal saline, placing the blood vessels into a normal saline solution containing 1% polystyrene, and preserving the blood vessels at a temperature of 4 ℃;
(2) Cell treatment
In an ultra-clean bench, cutting a blood vessel into 2-5 cm fragments, putting the fragments into 0.075% sodium dodecyl sulfate solution, shaking the solution gently at room temperature for 12 hours, flushing the solution with physiological saline for 3 times, and 4 hours each time to obtain extracellular matrix.
The method for preparing extracellular matrix by pig heart preparation or pig small intestine submucosa is as follows:
(1) Pretreatment of
Selecting a fresh pig heart front wall part or pig small intestine submucosa with no wound and uniform thickness, removing fat tissue, soaking the pig heart envelope or pig small intestine submucosa in deionized water and repeatedly washing for 3 times for 5 minutes each time, removing residual fat tissue with scissors, and trimming to about 10 x 10cm 2 Size, obtain pretreated material;
(2) Degreasing
Adjusting the temperature of the magnetic heating stirrer to 40 ℃, adding the magnetic heating stirrer into 0.4% sodium dodecyl sulfate according to the feed liquid ratio of 2.5-5 (preferably 3), and washing twice according to the feed liquid ratio of 3-6 (preferably 4) after rotating for 1.5h at the rotating speed of 120 r/min;
(3) Alkali treatment
Adjusting the temperature of the magnetic heating stirrer to 25-35 ℃, adding 0.2-1.4% (preferably 1.1%) NaOH solution according to the feed liquid ratio of 2.5-5 (preferably 3), adjusting the rotating speed to 120r/min, rotating for 30min, then rotating for 10min every 50min, rotating for 16h in total, and washing for three times according to the feed liquid ratio of 3-6 (preferably 4) after the treatment; it should be noted that, the alkaline solution has an ablation effect, can destroy cytoplasmic components, is favorable for removing partial immune components, and moreover, the fiber structure is seriously destroyed due to the overhigh temperature.
(4) Dealkalization: adjusting the temperature of the magnetic heating stirrer to 30 ℃, adding 3.0% ammonium chloride solution according to the feed-liquid ratio of 2.5-5 (preferably 3), adjusting the rotating speed to rotate for 1.5h, measuring the pH value once every 30min until the pH value reaches neutrality, and washing for three times according to the feed-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 0.2-0.5% of pancreatin solution or pepsin solution according to the feed-liquid ratio of 2-3 (preferably 2.5), adjusting the rotating speed to rotate for 30min, adding 0.2-0.8% of neutral protease solution or Triton-X-100 solution to rotate for 1-4 h after the completion, and washing 3 times according to the feed-liquid ratio of 2.5-5 (preferably 3);
(6) PBS wash: adjusting a magnetic heating stirrer to room temperature (25 ℃), and cleaning for 3 times according to a liquid ratio of 2.5-5 (preferably 3) for 15min each time to obtain an extracellular matrix;
(7) Sample preservation: the decellularized pericardium was stored in PBS buffer and placed in a refrigerator at 4 ℃.
In some embodiments of the invention, prior to filling the paraffin microspheres into the tubular space of the mold, further comprising:
immersing the mold in a polyethylene oxide solution;
the mold immersed in the polyethylene oxide solution is subjected to a drying treatment.
In this embodiment, the mold is immersed in the polyethylene oxide solution, so that the surface of the mold is covered with the polyethylene oxide solution, the mold is dried, and then the surface of the mold is covered with a layer of polyethylene oxide solid, the solid has a certain thickness and can occupy a certain space, and the space is a reserved space.
The thickness of the above-mentioned head space 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, immersing the mold in a polyethylene oxide solution comprises:
immersing a shell 1 included in a die in a polyethylene oxide solution with a concentration of 15-30 wt%;
immersing a column body 2 included in the die into a polyethylene oxide solution with the concentration of 8-12 wt%;
wherein the solvent of the polyethylene oxide solution is ethanol.
In this example, the shell 1 is immersed in a polyethylene oxide solution having a concentration of 15 to 30wt% to obtain a headspace having the same thickness as the extracellular matrix; the column 2 is immersed in polyethylene oxide solution with the concentration of 8-12 wt percent, so that the thin polyethylene oxide coated outside the column 2 can be obtained, and the thin polyethylene oxide is only used for facilitating the taking out of the paraffin network in the later period, so that the thickness is thinner, and the paraffin network can be conveniently demoulded after being washed.
It will be appreciated that the concentration of polyethylene oxide solution can be flexibly adjusted as required to accommodate extracellular matrices of varying thickness.
It should be noted that, selecting ethanol as solvent can shorten the time of removing solvent, and then shorten the preparation time of nerve conduit, in addition, ethanol is nontoxic after volatilizing, and will not pollute the environment and harm human health.
In some embodiments of the invention, the temperature of the heat treatment is 40-60 ℃ and the time of the heat treatment is 40-100 min.
In this embodiment, the temperature of the heating treatment is close to or equal to the melting point of paraffin, and the heating treatment is performed for 40-100 min at the heating temperature, so that the surface of the paraffin microsphere is melted, and the spherical morphology can be maintained, and thus adjacent paraffin microspheres are adhered to each other to form a paraffin network. If the temperature of the heating treatment is higher than 60 ℃ and/or the time of the heating treatment is longer than 100min, the paraffin microspheres cannot keep the spherical shape, and if the temperature of the heating treatment is lower than 40 ℃ and/or the time of the heating treatment is shorter than 40min, the paraffin microspheres cannot be melted, and cannot adhere to each other to form a paraffin network.
In some embodiments of the invention, after obtaining the paraffin network, before wrapping the extracellular matrix outside the paraffin network, further comprising:
immersing a mold containing a paraffin network in absolute ethyl alcohol;
taking the paraffin network out of the die;
cleaning and drying the die;
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 paraffin network is immersed in absolute ethanol, so that the paraffin network is conveniently taken out of the mold, and if polyethylene oxide is wrapped on the surface of the mold, the ethanol can dissolve the wrapped polyethylene oxide, so that the paraffin network is further conveniently taken out. The ethanol can also dissolve polyethylene oxide on the inner wall of the shell 1, so that space is provided for the extracellular matrix which is subsequently wrapped outside the paraffin network, and the paraffin network wrapping the extracellular matrix is conveniently filled into a die. Immersing the dried mould into polyethylene oxide solution with the concentration of 8-12 wt% again, taking out, setting the mould, and demoulding after immersing the mould in ethanol when the nerve conduit is required to be demoulded in the subsequent preparation.
In some embodiments of the invention, the bioprotein solution is a silk fibroin solution having a concentration of 10-15 wt%.
In this example, the 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 net-shaped solid obtained after freeze-drying is. If the concentration of the silk fibroin solution is higher than 15wt%, the specific surface area of the obtained net solid is low, and if the concentration of the silk fibroin solution is lower than 10wt%, the obtained net solid has poor supporting property.
The silk fibroin is a high-purity protein secreted by endothelial cells of silk glands at the rear part of silkworms, does not contain organelles and other biological impurities, and has high biological safety. The silk fibroin consists of twenty amino acids such as ethylamino acid, alanine and serine, has good biocompatibility, can be biodegraded, and the final degradation products are polypeptide and free amino acid which are easy to be metabolized by organisms. Studies have shown that silk fibroin materials are also well able to support the growth of neurons and neural stem cells. The catheter material prepared from the silk fibroin has low in-vivo inflammatory reaction and is degradable, and can promote the functional recovery of peripheral nerve defects.
In some embodiments of the invention, a method of preparing a silk fibroin comprises:
placing mulberry silk into 0.02mol/L Na at 100deg.C 2 CO 3 Boiling in solution twice for 30min each time, washing with tap water and distilled water for several times to thoroughly remove sericin, and air drying for use. Dissolving degummed silk in LiBr solution of 9.3mol/L, dissolving at 60deg.C for 4h, dialyzing for 3d (molecular weight cut-off is 12000-14000) to obtain silk solution with concentration of 8%, centrifuging at 12000rpm for 15min, concentrating low concentration PEG solution for a certain time to obtain high concentration silk solution, centrifuging at 12000rpm for 15min, centrifuging, and measuring concentration by dry constant weight method. Placing in a refrigerator at 4 ℃ for standby.
In some embodiments of the invention, the bioprotein solution is a collagen gel having a concentration of 0.8 to 1.4 wt%.
In this example, collagen has excellent biocompatibility, the degradation rate is matched with the tissue regeneration rate, and the higher the concentration of collagen gel is, the higher the density of the net-shaped solid obtained after freeze-drying is. If the concentration of the collagen gel is higher than 1.4wt%, the specific surface area of the obtained net-like solid is low, and if the concentration of the collagen gel is lower than 10wt%, the obtained net-like solid has poor supporting property.
In some embodiments of the invention, the method of preparing a collagen gel comprises:
(1) Removing superfluous fascia, fat, muscle and the like on the beef achilles tendon, washing with tap water, and orderly arranging in a freezing box for freezing at-20 ℃ for at least 12 hours;
(2) Cutting frozen Achilles tendon into slices of about 1mm, and placing into a filter screen for turning and washing until the liquid is clear;
(3) Enzymolysis: carrying out enzymolysis on the cleaned beef achilles tendon slices, and fully stirring, wherein the enzymolysis time is not less than 72 hours; 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, separating out white flocculent collagen, filtering and cleaning, and draining water.
(5) And (3) dialysis: filling the salted-out material into a dialysis bag, wherein the filling volume is about 1/3 of that of the dialysis bag; placing the dialysis bag in 0.057mol/L acetic acid solution for 6 days, and changing the dialysis solution every 3 days at 10-20deg.C; 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 changed every 1 day; dialyzing in 0.0000057mol/L acetic acid solution from day 12 to pH 5.0-5.5, and dialyzing at 10-20deg.C, and changing dialysate once daily as required.
(6) The collagen gel was removed and tested for solids content.
In some embodiments of the invention, lyophilizing and removing paraffin network from a solid-liquid mixture comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
immersing the freeze-dried tube body in 90wt% methanol for 2 hours;
the paraffin network in the freeze-dried tube is washed off with n-hexane.
In this example, soaking the freeze-dried tube in 90wt% methanol for 2 hours allowed the silk fibroin to set, increasing the support of the resulting reticulated solid. In addition, the method of washing off the paraffin network may be a Soxhlet extraction method, specifically, the paraffin network inside the stent is washed off with n-hexane, then n-hexane is replaced with cyclohexane, and then the cyclohexane is removed by vacuum.
In some embodiments of the invention, the lyophilization process includes a pre-freezing stage, a first sublimation stage, a second sublimation stage, and a cooling stage, the process conditions for each stage being as follows:
pre-freezing: the target temperature is-12 to-8 ℃, the speed is 3-4.0 ℃/min, and the constant temperature duration is 280-320 min;
a first sublimation stage: vacuumizing, aerating at 90-110 Pa, wherein the target temperature is-4 to-2 ℃, the speed is 0.6-0.8 ℃/min, and the constant temperature duration is 1300-1340 min;
in the second sublimation stage, vacuumizing, and aerating at 90-110 Pa, wherein the second sublimation stage comprises five temperature rising steps of respectively:
-1 ℃, the speed is 0.2-0.3 ℃/min, and the constant temperature duration is 110-130 min;
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;
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, and the constant temperature duration is as follows: performing end point judgment every 10 minutes until the end point judgment is qualified; judging the end point to be less than or equal to 0.9Pa/10min;
and (3) a cooling stage: cooling to room temperature at a speed of 1.4-1.6 ℃/min.
In this embodiment, the pre-cooling stage may be performed by treating at-20deg.C for 10-14 h, at-70deg.C for 2-6 h or in liquid nitrogen for 3-10 min, and freeze-drying the rest steps according to the above process.
In some embodiments of the invention, lyophilizing and removing paraffin network from a solid-liquid mixture comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
crosslinking the freeze-dried pipe body;
the paraffin network in the freeze-dried tube is washed off with n-hexane.
In this embodiment, the cross-linking treatment of the freeze-dried tube body can avoid the swelling of collagen when contacting water to damage the structure of the mesh solid, and increase the stability of the prepared mesh solid. In addition, the method of washing off the paraffin network may be a Soxhlet extraction method, specifically, the paraffin network inside the stent is washed off with n-hexane, then n-hexane is replaced with cyclohexane, and then the cyclohexane is removed by vacuum.
In some embodiments of the invention, the process of crosslinking treatment comprises:
a. preparing a cross-linking agent solution: the edible alcohol with a certain volume is measured by a measuring cylinder and is transferred into a reaction kettle, glutaraldehyde solution with a certain volume (the mass fraction of glutaraldehyde is 50%) is transferred by a liquid transferring gun, and the volume ratio of the edible alcohol to the glutaraldehyde solution is 100: preparing glutaraldehyde alcohol solution in a proportion of 0.05;
b. the material is soaked in the crosslinking liquid, the stirring speed of the reaction kettle is 20-30r/min, and the uppermost material can rotate along with stirring and is crosslinked for 48h. (Note: when crosslinking, after the material adsorbed the crosslinking agent and sunk, if the material is below the stirring paddle, the stirring paddle is not in contact with the material, the stirring speed is reduced, instead 10r/min. If the stirring paddle is in contact with the material, the stirring is turned off).
c. Washing process
(1) Taking the crosslinked material out of the crosslinking agent solution;
(2) The centrifuge loads the filter bag and centrifuges once (5 seconds); the rotational speed of the centrifugal machine defaults to 3000r/min;
(3) Soaking with 3% hydrogen peroxide solution for 42+ -1 h at a ratio of 1:80, i.e. 3% hydrogen peroxide solution volume (ml) =product mass (g) ×80, the product being completely immersed in hydrogen peroxide solution.
(4) Performing ultrasonic treatment on the purified water for 30min, and centrifuging once (5 s) after ultrasonic treatment; repeating for 2 times;
(5) The edible alcohol is treated by ultrasonic for 5min, and the volume of the edible alcohol is based on the principle of immersing the product. After the ultrasonic treatment is finished, the centrifugal operation is carried out once (5 seconds);
d. vacuum drying
And (3) loading 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 invention, after obtaining the nerve conduit, further comprising:
injecting 2-10wt% concentration egg yolk lecithin solution into the inner layer structure;
the nerve conduit injected with the egg yolk lecithin solution was subjected to vacuum drying treatment.
In this example, egg yolk lecithin is digested to release choline, and choline and acetyl coenzyme are catalyzed by choline acetyltransferase to react to form acetylcholine, which is the chemical transmitter of all synapses, muscle nerve joints, all parasympathetic postganglionic fibers and postganglionic fibers of sympathetic nerves between the pre-ganglionic and postganglionic fibers of the autonomic nervous system, and the increase of the content of the acetylcholine can promote the rapid enhancement of brain nerve synapses, so that the information transmission speed between nerve cells in the brain is accelerated, and the memory and learning ability of a person are improved. In addition, egg yolk lecithin can be used for treating various neurosis. If the mass fraction of the egg yolk lecithin is less than 2wt%, the effect of repairing nerves is poor, and if the mass fraction of the egg yolk lecithin is more than 10wt%, the porosity of the network structure is reduced.
The egg yolk lecithin solution may be injected into the net structure by a syringe.
As shown in fig. 2, the present invention further provides another method for preparing a nerve conduit, which includes:
step 200, immersing the shell 1 included in the die into a polyethylene oxide solution with the concentration of 15-30wt%;
step 202, immersing a column body 2 included in a die into a polyethylene oxide solution with the concentration of 8-12 wt%;
step 204, drying the mould immersed in the polyethylene oxide solution;
step 206, filling paraffin microspheres into the tubular space of the mold; the die comprises a shell 1 and a column body 2, wherein the inner wall of the shell is columnar, the column body 2 is arranged in the shell 1, and a tubular space is formed between the column body 2 and the shell 1;
step 208, heating the paraffin microspheres to obtain a paraffin network;
step 210, immersing a mould containing a paraffin network into absolute ethyl alcohol;
step 212, taking the paraffin network out of the mold;
step 214, cleaning and drying the mold;
step 216, immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt% and then taking out;
step 218, wrapping the extracellular matrix outside the paraffin network to obtain an outer layer structure;
step 220, injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture;
step 222, carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
step 224, soaking the freeze-dried tube body in 90wt% methanol for 2 hours;
step 226, cleaning the paraffin network in the freeze-dried tube body by using normal hexane, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with a double-layer composite structure;
step 228, injecting a yolk lecithin solution with the concentration of 2-10wt% into the inner layer structure;
at step 230, the nerve conduit infused with egg yolk lecithin solution is subjected to a vacuum drying process.
As shown in fig. 3, the embodiment of the present invention further provides a preparation method of a nerve conduit, which includes:
step 300, immersing the shell 1 included in the die into a polyethylene oxide solution with the concentration of 15-30wt%;
step 302, immersing a column 2 included in the die into a polyethylene oxide solution with a concentration of 8-12 wt%;
step 304, drying the mould immersed in the polyethylene oxide solution;
step 306, filling paraffin microspheres into the tubular space of the mold; the die comprises a shell 1 and a column body 2, wherein the inner wall of the shell is columnar, the column body 2 is arranged in the shell 1, and a tubular space is formed between the column body 2 and the shell 1;
step 308, heating the paraffin microspheres to obtain a paraffin network;
step 310, immersing a mould containing a paraffin network into absolute ethyl alcohol;
step 312, taking the paraffin network out of the mold;
step 314, cleaning and drying the mold;
step 316, immersing the die into a polyethylene oxide solution with the concentration of 8-12 wt% and then taking out;
step 318, wrapping the extracellular matrix outside the paraffin network to obtain an outer layer structure;
step 320, injecting a biological protein solution into the paraffin network space wrapped by the outer layer structure to obtain a solid-liquid mixture;
step 322, performing freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
step 324, performing cross-linking treatment on the freeze-dried pipe body;
step 326, cleaning the paraffin network in the freeze-dried tube body by using normal hexane, and obtaining an inner layer structure inside the outer layer structure to obtain the nerve conduit with a double-layer composite structure;
328, injecting a yolk lecithin solution with the concentration of 2-10wt% into the inner layer structure;
and 330, performing vacuum drying treatment on the nerve conduit injected with the egg yolk lecithin solution.
In order to more clearly illustrate the technical scheme and advantages of the present invention, a method for preparing a nerve conduit will be described in detail below through several examples.
Example 1
(1) Mold pretreatment
Immersing the shell and the column body of the mould into polyethylene oxide (molecular weight 2000) solution with concentration of 20wt% and concentration of 10wt%, respectively, taking out and drying; wherein the solvent is ethanol;
(2) Preparation of Paraffin networks
Filling paraffin microspheres into the die treated in the step (1), gently oscillating, and heating at 50 ℃ for 60min to obtain a paraffin network;
(3) Encapsulation of extracellular matrix
Immersing the mould in ethanol, slightly taking out the paraffin network, and wrapping the extracellular matrix outside the paraffin network to obtain an outer layer structure;
(4) Preparation of nerve conduit
Cleaning and drying a mould, placing the outer layer structure and a paraffin network wrapped by the outer layer structure in the mould, injecting a silk fibroin solution with the concentration of 13wt%, carrying out freeze-drying treatment to obtain a freeze-dried body, soaking the freeze-dried body in methanol with the concentration of 90% for 2 hours, and removing the paraffin network in the freeze-dried body to obtain the nerve conduit;
(5) Composite egg yolk lecithin
And injecting a yolk lecithin solution with the concentration of 6wt% into the inner layer structure, filling the yolk lecithin solution into the gaps of the net 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 substantially the same as example 1 except that:
in step (1), immersing the shell and the column comprised by the mold in polyethylene oxide (molecular weight 2000) solutions having a concentration of 15wt% and a concentration of 10wt%, respectively;
in the step (2), heating at 40 ℃ for 40min;
in step (4), a silk fibroin solution with a concentration of 10wt% is injected;
in step (5), a 2wt% strength egg yolk lecithin solution was injected into the inner layer structure.
Example 3
Example 3 is substantially the same as example 1 except that:
in step (1), immersing the shell and the column comprised by the mold in a polyethylene oxide (molecular weight 2000) solution having a concentration of 30wt% and a concentration of 12wt%, respectively;
in the step (2), heat treatment is carried out at 60 ℃ for 100min;
in step (4), a silk fibroin solution with a concentration of 15wt% is injected;
in step (5), a 10wt% strength egg yolk lecithin solution is injected into the inner layer structure.
Example 4
Example 4 is substantially the same as example 1 except that:
in the step (4), the mold is cleaned and dried, the outer layer structure and the paraffin network wrapped by the outer layer structure are placed in the mold, collagen gel with the concentration of 1.2wt% is injected, the freeze-drying treatment is carried out to obtain a freeze-dried body, the cross-linking treatment is carried out on the freeze-dried body, and the paraffin network in the freeze-dried body is removed to obtain the nerve conduit.
Example 5
Example 5 is substantially the same as example 2 except that:
in the step (4), the mold is cleaned and dried, the outer layer structure and the paraffin network wrapped by the outer layer structure are placed in the mold, collagen gel with the concentration of 0.8wt% is injected, the freeze-drying treatment is carried out to obtain a freeze-dried body, the cross-linking treatment is carried out on the freeze-dried body, and the paraffin network in the freeze-dried body is removed to obtain the nerve conduit.
Example 6
Example 6 is substantially the same as example 3 except that:
in the step (4), the mold is cleaned and dried, the outer layer structure and the paraffin network wrapped by the outer layer structure are placed in the mold, collagen gel with the concentration of 1.4wt% is injected, the freeze-drying treatment is carried out to obtain a freeze-dried body, the cross-linking treatment is carried out on the freeze-dried body, and the paraffin network in the freeze-dried body is removed to obtain the nerve conduit.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (5)
1. A method of preparing a nerve conduit, comprising:
filling paraffin microspheres into a tubular space of a mold; the die comprises a shell with a columnar inner wall and a column body, wherein the column body is arranged in the shell, and a tubular space is formed between the column body and the shell;
heating the paraffin microspheres to obtain a paraffin network;
wrapping an 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;
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 a double-layer composite structure;
before the paraffin microspheres are filled into the tubular space of the die, the method further comprises:
immersing the mold in a polyethylene oxide solution;
drying the mould immersed in the polyethylene oxide solution;
the immersing the mold in a polyethylene oxide solution comprises:
immersing a shell of the die into polyethylene oxide solution with the concentration of 15-30wt%;
immersing a column body included in a die into a polyethylene oxide solution with the concentration of 8-12wt%;
wherein the solvent of the polyethylene oxide solution is ethanol;
the temperature of the heating treatment is 40-60 ℃, and the time of the heating treatment is 40-100 min;
after the obtaining of the paraffin network, before the encapsulating of the extracellular matrix outside the paraffin network, further comprising:
immersing the mold containing the paraffin network in absolute ethanol;
removing the paraffin network from the mould;
cleaning and drying the die;
immersing the die into a polyethylene oxide solution with the concentration of 8-12wt% and taking out;
after obtaining the nerve conduit, the method further comprises:
injecting a yolk lecithin solution with the concentration of 2-10wt% into the inner layer structure;
vacuum drying the nerve conduit injected with the egg yolk lecithin solution;
the porosity of the inner layer structure is 70-85%, and the specific surface area is 15-21 m 2 /g。
2. The preparation method of claim 1, wherein the biological protein solution is a silk fibroin solution with a concentration of 10-15 wt%.
3. The method according to claim 1, wherein the bioprotein solution is collagen gel having a concentration of 0.8-1.4 wt%.
4. The method according to claim 2, 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;
immersing the freeze-dried tube body in 90wt% methanol for 2 hours;
and cleaning the paraffin wax network in the freeze-dried tube body by using n-hexane.
5. A method of preparing according to claim 3, wherein said lyophilizing said solid-liquid mixture and removing said paraffin network comprises:
carrying out freeze-drying treatment on the solid-liquid mixture to obtain a freeze-dried pipe body;
carrying out cross-linking treatment on the freeze-dried pipe body;
and cleaning the paraffin wax network in the freeze-dried tube body by using n-hexane.
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| CN116531143B (en) * | 2023-07-07 | 2023-11-10 | 天新福（北京）医疗器材股份有限公司 | Method for improving uniformity of double-layer freeze-dried tubular substance and application thereof |
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| US20040028738A1 (en) * | 2000-10-05 | 2004-02-12 | Lynn L.H. Huang | Process for the preparation of porous collagen matrix |
| JP4638735B2 (en) * | 2002-06-24 | 2011-02-23 | タフツ ユニバーシティー | Silk biomaterial and method of using the same |
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| WO2001003609A1 (en) * | 1999-07-07 | 2001-01-18 | Tapic International Co., Ltd. | Artificial neural tube |
| CN102178980A (en) * | 2011-05-05 | 2011-09-14 | 东南大学 | Natural polymer composite porous fibrous scaffold and preparation method thereof |
| CN104399131A (en) * | 2014-10-21 | 2015-03-11 | 赵红斌 | Multi-channel nerve repair conduit with tissue induced function and mold |
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