CN116139335B - Multichannel aperture gradient hydrogel stent with directional conveying capability and preparation method thereof - Google Patents
Multichannel aperture gradient hydrogel stent with directional conveying capability and preparation method thereof Download PDFInfo
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 54
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 108010022355 Fibroins Proteins 0.000 claims abstract description 49
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 47
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 23
- 239000011148 porous material Substances 0.000 claims abstract description 20
- 230000009471 action Effects 0.000 claims abstract description 9
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 72
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- 238000003756 stirring Methods 0.000 claims description 23
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- 239000007864 aqueous solution Substances 0.000 claims description 10
- 239000000835 fiber Substances 0.000 claims description 8
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 230000008859 change Effects 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 5
- 241000255789 Bombyx mori Species 0.000 claims description 5
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 5
- 239000011837 N,N-methylenebisacrylamide Substances 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 5
- 238000009835 boiling Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 claims description 5
- 229920002401 polyacrylamide Polymers 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
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- 230000007423 decrease Effects 0.000 claims description 2
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- 230000001105 regulatory effect Effects 0.000 claims description 2
- 230000003247 decreasing effect Effects 0.000 claims 1
- 210000005036 nerve Anatomy 0.000 abstract description 33
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 abstract description 15
- 230000008929 regeneration Effects 0.000 abstract description 12
- 238000011069 regeneration method Methods 0.000 abstract description 12
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 6
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- 208000028389 Nerve injury Diseases 0.000 description 1
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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Abstract
The multi-channel pore-size gradual-change hydrogel bracket with directional conveying capability and the preparation method thereof are provided, wherein the bracket is polyacrylamide-silk fibroin-polyethylene glycol hydrogel and has a plurality of pore-size gradual-change porous micro-channel structures, and the pore size is gradually reduced from 1000-600 mu m to 100-200 mu m; the preparation method comprises the following steps: firstly, synthesizing acrylamide and silk fibroin into acrylamide-silk fibroin through dehydration condensation reaction; and synthesizing polyacrylamide-silk fibroin-polyethylene glycol (PAM-SF-PEG) hydrogel by esterification reaction of the acrylamide-silk fibroin and polyethylene glycol; then filling PAM-SF-PEG hydrogel into a specific mold to prepare a porous micro-channel pore-diameter gradient hydrogel bracket with bionic plant capillary action; the hydrogel bracket is implanted into a long-distance peripheral nerve defect part, so that good mechanical support can be provided for nerve regeneration, the porous micro-channel structure of the hydrogel bracket can guide the directional growth of a new nerve axon, promote the accuracy of nerve involution and prevent nerve mispairing.
Description
Technical Field
The invention relates to the technical field of high polymer materials, in particular to a multichannel pore-size gradient hydrogel bracket with directional conveying capacity and a preparation method thereof.
Background
Peripheral nerve injury is one of the common clinical diseases and accounts for 2% -5% of all wound cases. The peripheral nerves of people are damaged to different degrees due to various factors such as natural aging, iatrogenic side effects, accidents and the like, so that the nerve signal transmission is interrupted and the functions of corresponding target organs are damaged, and further, the sensory and motor functions are lost, and muscle paralysis and even life-long disability are caused. Repair and regeneration of peripheral nerve damage is therefore of great importance to the life health and medical system of the patient.
The end-to-end stitching adopted for clinically repairing peripheral nerve injury is difficult to repair long-distance nerve defects, and the problems of shortage of donor nerves, poor matching performance, easiness in forming postoperative neuroma and the like exist in autograft. The hydrogel bracket provides a new choice for repairing peripheral nerve injury, the three-dimensional structure of the hydrogel bracket can provide a proper microenvironment for the growth and functionalization of nerve cells, the surface structure of the hydrogel bracket can guide the directional growth of new nerve axons, the nerve involution accuracy is promoted, meanwhile, the hydrogel bracket can provide enough mechanical support for nerve regeneration, the tension of surgical suture is relieved, and the nerve regeneration is effectively promoted. However, although various hydrogel scaffolds have been applied to repair of peripheral nerve injury, such as the name Electrically Conductive Hydrogel Nerve Guidance Conduits forPeripheralNerve Regeneration (conductive hydrogel nerve guiding catheter for regeneration of peripheral nerve) published in Adv FunctMaterials, these conductive hydrogel nerve guiding catheters cannot effectively support repair and regeneration of the distal end of the injured nerve due to gravity, and have problems of unsatisfactory long-distance nerve defect repair effect, long postoperative recovery time, and the like.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a multichannel aperture gradual change hydrogel bracket with directional conveying capability and a preparation method thereof, and the invention firstly synthesizes Acrylamide (AM) and Silk Fibroin (SF) into Acrylamide-Silk fibroin (AM-SF) through dehydration condensation reaction; and synthesizing Polyacrylamide (PAM) -silk fibroin-polyethylene glycol (PAM-SF-PEG) hydrogel by esterification reaction of the acrylamide-silk fibroin and polyethylene glycol (Polyethylene glycol, PEG); and then filling the PAM-SF-PEG hydrogel into a specific mould to prepare the porous microchannel hydrogel bracket with bionic plant capillary action, which can be used for directionally transferring cells against gravity.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the multichannel pore-size gradual change hydrogel bracket with directional conveying capability is polyacrylamide-silk fibroin-polyethylene glycol hydrogel, has a plurality of pore-size gradual change porous micro-channel structures, and gradually reduces the pore size from 1000-600 mu m to 100-200 mu m.
The polyacrylamide-silk fibroin-polyethylene glycol hydrogel is as follows: 40-60 wt% of polyacrylamide solution, 5-10wt% of silk fibroin solution and 8-10wt% of polyethylene glycol solution, and mixing the three solutions in equal volume ratio.
The preparation method of the multichannel aperture gradient hydrogel stent with directional conveying capability comprises the following steps:
step one: firstly, cutting silkworm cocoons into slices, boiling with 0.5wt% of Na2CO3 aqueous solution for 2-3 times, washing with deionized water, and drying to obtain degummed silk fibroin fibers; dissolving with ternary solution or 5-8wt% lithium bromide/methanol solution, centrifuging to remove impurities, separating out the centrifuged silk fibroin solution, and freeze-drying to obtain solid silk fibroin SF;
step two: firstly, dissolving AM in deionized water to obtain 40-60 wt% solution, adding 0.4-0.6 wt% N, N-methylene bisacrylamide, and stirring at normal temperature to obtain AM solution; then SF is dissolved in deionized water to obtain SF water solution with the concentration of 5-10wt%, and the SF water solution is uniformly stirred, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are respectively added, the addition amounts are 0.5-2wt%, the PH is regulated to 4.5-5.5, and the SF water solution is obtained after the SF water solution reacts for 1-2 hours at room temperature; preparing 8-10wt% polyethylene glycol PEG with deionized water, and stirring at normal temperature to prepare PEG solution;
step three: mixing the AM solution, the SF aqueous solution and the PEG solution in the second step according to the volume ratio of 1:1:1, stirring and reacting to obtain the AM-SF-PEG solution, then adding 0.1-0.2 wt% of ammonium persulfate and 0.08-0.16 wt% of tetramethyl ethylenediamine, stirring and mixing uniformly, adding into a mould, and reacting for 10-15 hours at 50-70 ℃ to obtain the PAM-SF-PEG hydrogel with the bionic capillary action.
The ternary solution is anhydrous calcium chloride/water/absolute ethyl alcohol, and the volume ratio of the ternary solution to the water/absolute ethyl alcohol is 1:8:2.
The die is of a cylindrical structure, a plurality of pore diameter gradual change porous micro-channel structures are arranged in the height direction of the cylinder, and the diameters of the pores are gradually reduced from 1000-600 mu m to 100-200 mu m.
Compared with the prior art, the invention has the advantages that:
1. after the hydrogel stent is implanted into a long-distance peripheral nerve defect, the porous micro-channel structure can guide the directional growth of a new nerve axon, promote the accuracy of nerve involution and prevent nerve mispairing; the pore-diameter gradual-change porous micro-channel structure can be used for conveying nerve cells to the far end of the nerve defect, further promoting the regeneration of the far-end nerve, improving the long-distance nerve defect repair efficiency and shortening the recovery period of a patient.
2. The polyacrylamide-silk fibroin-polyethylene glycol hydrogel prepared by the method has good mechanical properties, strain reaches more than 100%, and has good flexibility; the stress reaches more than 50Kpa and even exceeds 100Kpa, so that sufficient mechanical support is provided for nerve regeneration, and the tension of surgical suture is reduced.
Drawings
FIG. 1 is a schematic view of a hydrogel scaffold with a pore size gradient porous microchannel structure according to the present invention.
FIG. 2 is a graph of mechanical properties of hydrogels of three embodiments of the present invention.
FIG. 3 is a graph of the microscopic morphology of pore size progression of AM-SF-PEG-2 hydrogels for second biomimetic capillary action in accordance with an embodiment of the present invention.
Detailed Description
The present invention will be described in more detail with reference to examples.
The multichannel pore size gradient hydrogel scaffold with directional conveying capability is polyacrylamide-silk fibroin-polyethylene glycol hydrogel, and the polyacrylamide-silk fibroin-polyethylene glycol hydrogel is as follows: 40-60 wt% of polyacrylamide solution, 5-10wt% of silk fibroin solution and 8-10wt% of polyethylene glycol solution, and the three solutions are mixed in equal proportion.
The multichannel pore-size gradual-change hydrogel bracket with directional conveying capability is provided with a plurality of pore-size gradual-change porous micro-channel structures, as shown in fig. 1 and 3, wherein the pore size gradually decreases from 1000-600 mu m to 100-200 mu m. The hydrogel scaffold has about 30 micro-channel structures, and the pore size gradient hydrogel scaffold can be used for antigravity directional cell transport; after the hydrogel stent is implanted into a long-distance peripheral nerve defect, the porous micro-channel structure can guide the directional growth of a new nerve axon, promote the accuracy of nerve involution and prevent nerve mispairing; the pore-diameter gradual-change porous micro-channel structure can be used for conveying nerve cells to the far end of the nerve defect, so that the long-distance nerve defect repairing efficiency is improved, and the recovery period of a patient is shortened.
Example 1
The embodiment comprises the following steps:
step one: firstly, cutting silkworm cocoons into slices, using 0.5% of Na 2 CO 3 Boiling with water solution (bath ratio of 1:50) for 2 times (30 minutes each time), washing with deionized water for 3 times, and drying to obtain degummed silk fibroin fiber. 100g of degummed silk fibroin fiber was then dissolved in 1000mL of ternary solution (anhydrous calcium chloride/water/anhydrous ethanol in a volume ratio of 1:8:2) and completely dissolved in a water bath at 70℃and centrifuged in a centrifuge to remove impurities. Finally, dialyzing the centrifuged silk fibroin solution for 72 hours, freeze-drying for 48 hours to obtain solid silk fibroin SF, and storing the solid silk fibroin SF in a refrigerator at 4 ℃ for later use;
step two: firstly, dissolving AM in deionized water to obtain 40wt% solution, adding 0.4wt% of N, N-methylene bisacrylamide, and stirring for 30 minutes at normal temperature to obtain AM solution. SF was then dissolved in 5mL of deionized water to give a 5wt% SF aqueous solution, and a magnetic stirrer was used for 30 minutes, and 0.5wt% 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 0.5wt% N-hydroxysuccinimide were added, respectively, and the pH was adjusted to 5, and reacted at room temperature for 1 hour to give SF solution. Preparing 5mL of 5% (w/v) PEG with deionized water, and stirring at normal temperature for 10 minutes to prepare a polyethylene glycol PEG solution;
step three: mixing the AM solution, the SF aqueous solution and the PEG solution in the second step according to the volume ratio of 1:1:1, stirring and reacting for 1 hour to obtain 15mLAM-SF-PEG solution, then adding 0.1wt% ammonium persulfate and 0.08wt% tetramethyl ethylenediamine, stirring and mixing uniformly, adding a die, and reacting for 12 hours at the temperature of 60 ℃ to obtain the AM-SF-PEG-1 (ASP-1) hydrogel with the bionic capillary action.
Example two
The embodiment comprises the following steps:
step one: firstly, cutting silkworm cocoons into slices, boiling the slices 3 times (30 minutes each time) by using 0.5% Na2CO3 aqueous solution (bath ratio is 1:50), washing the slices by using deionized water 3 times, and drying the slices to obtain degummed silk fibroin fibers. 100g of degummed silk fibroin fibers were then dissolved in 1000mL of a 5wt% lithium bromide/methanol solution and completely dissolved in a 70℃water bath, and centrifuged in a centrifuge to remove impurities. Finally, dialyzing the centrifuged silk fibroin solution for 72 hours, freeze-drying for 48 hours to obtain solid silk fibroin, and storing the solid silk fibroin in a refrigerator at 4 ℃ for later use;
step two: firstly, dissolving AM in deionized water to obtain 50wt% solution, adding 0.5wt% of N, N-methylene bisacrylamide, and stirring for 30 minutes at normal temperature to obtain AM solution; SF was then dissolved in deionized water to give a 5wt% SF aqueous solution, and a magnetic stirrer was used for 30 minutes, and 1wt% 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1wt% N-hydroxysuccinimide were added, respectively, and the pH was adjusted to 4.5, and reacted at room temperature for 1 hour to give SF solution. Preparing 8wt% polyethylene glycol PEG with deionized water, and stirring at normal temperature to prepare a PEG solution;
step three: mixing the AM solution, the SF aqueous solution and the PEG solution in the second step according to the volume ratio of 1:1:1, stirring and reacting for 1 hour, stirring and reacting to obtain the AM-SF-PEG solution, adding 0.15wt% of ammonium persulfate and 0.12wt% of tetramethyl ethylenediamine into the AM-SF-PEG mixed solution, stirring and mixing uniformly, adding a mould, and reacting for 10 hours at 50 ℃ to obtain the AM-SF-PEG-2 (ASP-2) hydrogel with the bionic capillary action, wherein the pore diameter gradually becomes smaller from 600 mu m to 200 mu m, and the pore diameter gradually changes as can be seen from figure 3.
Example III
The embodiment comprises the following steps:
step one: firstly, cutting silkworm cocoons into slices, boiling the slices 3 times (30 minutes each time) by using 0.5 weight percent of Na2CO3 aqueous solution (bath ratio is 1:50), washing the slices by using deionized water 3 times, and drying the slices to obtain the degummed silk fibroin fibers. 100g of degummed silk fibroin fibers were then dissolved in 1000mL of an 8wt% lithium bromide/methanol solution and completely dissolved in a water bath at 70℃and centrifuged in a centrifuge to remove impurities. Finally, dialyzing the centrifuged silk fibroin solution for 72 hours, freeze-drying for 48 hours to obtain solid silk fibroin, and storing the solid silk fibroin in a refrigerator at 4 ℃ for later use;
step two: firstly, dissolving AM in deionized water to obtain 60wt% solution, adding 0.6wt% of N, N-methylene bisacrylamide, and stirring for 30 minutes at normal temperature to obtain AM solution; then SF is dissolved in 5mL of deionized water to obtain 10wt% SF water solution, a magnetic stirrer is used for 30 minutes, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are respectively added, the addition amounts are 2wt%, the PH is adjusted to 5.5, and the reaction is carried out at room temperature for 2 hours to obtain SF solution; preparing 8wt% polyethylene glycol PEG with deionized water, and stirring at normal temperature to prepare PEG solution.
Step three: mixing the AM solution, the SF aqueous solution and the PEG solution in the second step according to the volume ratio of 1:1:1, stirring and reacting to obtain an AM-SF-PEG solution, stirring and reacting for 1 hour, adding 0.2wt% of ammonium persulfate and 0.16wt% of tetramethyl ethylenediamine into the AM-SF-PEG mixed solution, stirring and mixing uniformly, adding into a mould, and reacting for 12 hours at the temperature of 60 ℃ to obtain the AM-SF-PEG-3 (ASP-3) hydrogel with the bionic capillary action.
As shown in figure 2, the polyacrylamide-silk fibroin-polyethylene glycol hydrogel prepared by the invention has good mechanical properties, strain reaches more than 100%, and has good flexibility; the stress reaches more than 50Kpa and even exceeds 100Kpa, so that sufficient mechanical support is provided for nerve regeneration, and the tension of surgical suture is reduced. In the nerve regeneration process, the far and near ends of the damaged nerve can be automatically identified, the nerve tissue grows from the near end to the far end and selectively extends to the corresponding target organ, and the hydrogel bracket can convey nerve cells to the far end of the nerve defect by utilizing capillary action, so that the nerve regeneration is further promoted, and the recovery period of a patient is shortened.
Claims (6)
1. The multichannel pore-size gradual change hydrogel bracket with directional conveying capability is characterized by being polyacrylamide-silk fibroin-polyethylene glycol hydrogel and having a plurality of pore-size gradual change porous micro-channel structures, wherein the pore size gradually decreases from 1000-600 mu m to 100-200 mu m.
2. The multi-channel pore size gradient hydrogel scaffold with directional delivery capability of claim 1, which is a polyacrylamide-silk fibroin-polyethylene glycol hydrogel with a number of pore size gradient porous micro-channel structures, with pore size gradually decreasing from 600 μm to 200 μm.
3. The multi-channel pore size gradient hydrogel scaffold with directional delivery capability of claim 1 or 2, wherein the polyacrylamide-silk fibroin-polyethylene glycol hydrogel is: 40-60 wt% polyacrylamide solution, 5-10wt% silk fibroin solution and 8-10wt% polyethylene glycol solution, and the three solutions are mixed in equal volume ratio.
4. The preparation method of the multichannel aperture gradient hydrogel stent with directional conveying capability comprises the following steps:
step one: firstly, cutting silkworm cocoons into slices, using Na 2 CO 3 Boiling for 2-3 times with water solution, washing with deionized water, and drying to obtain degummed silk fibroin fiber; dissolving with ternary solution anhydrous calcium chloride/water/anhydrous ethanol or lithium bromide/methanol solution, centrifuging to remove impurities, separating out the centrifuged silk fibroin solution, and freeze-drying to obtain solid silk fibroin SF;
step two: firstly, dissolving AM in deionized water to obtain 40-60 wt% solution, adding 0.4-0.6 wt% N, N-methylene bisacrylamide, and stirring at normal temperature to obtain AM solution; then SF is dissolved in deionized water to obtain SF water solution with the concentration of 5-10wt%, and the SF water solution is uniformly stirred, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are respectively added, the addition amounts are 0.5-2wt%, the pH is regulated to 4.5-5.5, and the SF water solution is obtained after the SF water solution reacts for 1-2 hours at room temperature; preparing 8-10wt% polyethylene glycol PEG with deionized water, and stirring at normal temperature to prepare PEG solution;
step three: mixing the AM solution, the SF aqueous solution and the PEG solution in the second step according to the volume ratio of 1:1:1, stirring and reacting to obtain the AM-SF-PEG solution, then adding 0.1-0.2 wt% of ammonium persulfate and 0.08-0.16 wt% of tetramethyl ethylenediamine, stirring and mixing uniformly, adding a mould after stirring and mixing uniformly, and reacting for 10-15 hours at 50-70 ℃ to obtain the PAM-SF-PEG hydrogel with bionic capillary action.
5. The method for preparing the multichannel pore size gradient hydrogel scaffold with directional delivery capability according to claim 4, comprising the following steps: the die is of a cylindrical structure, a plurality of pore diameter gradual change porous micro-channel structures are arranged in the height direction of the cylinder, and the diameters of the pores are gradually reduced from 1000-600 mu m to 100-200 mu m.
6. The method for preparing the multichannel pore size gradient hydrogel scaffold with directional delivery capability according to claim 4, comprising the following steps: the ternary solution is anhydrous calcium chloride/water/absolute ethyl alcohol, and the volume ratio of the ternary solution to the water/absolute ethyl alcohol is 1:8:2.
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| ES2405789T3 (en) * | 2010-04-15 | 2013-06-03 | National University Of Ireland | Multichannel collagen nerve conduit for nerve repair |
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