CN114470325A

CN114470325A - Preparation method of chitin hydrogel nerve conduit material capable of being sewn and cut into single-sided conductive and antibacterial material

Info

Publication number: CN114470325A
Application number: CN202210296241.XA
Authority: CN
Inventors: 王慧庆
Original assignee: Hefei University of Technology
Current assignee: Hefei University of Technology
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-05-13

Abstract

The invention provides a preparation method of a single-sided conductive nano polyaniline chitin nerve conduit material, belonging to the technical field of biological medicines. The operation steps are as follows: adding pure chitin powder into an alkali urea aqueous solution, freezing, thawing, stirring and alternately repeating the operation for three times until the pure chitin powder is completely dissolved to obtain a colorless and transparent chitin solution; laying on a glass plate mould, standing in a low-temperature coagulating bath, and slowly completing gelation to obtain the chitin network hydrogel film; placing the chitin network hydrogel film in the middle of a U-shaped reactor, wherein one side of the chitin network hydrogel film is a conductive high-molecular monomer organic solution, the other side of the chitin network hydrogel film is an initiator phytic acid aqueous solution, and carrying out ice-bath reaction to obtain a single-side conductive chitin hydrogel nerve conduit material, wherein one side of the single-side conductive chitin hydrogel nerve conduit material is highly transparent chitin hydrogel, the other side of the single-side conductive chitin hydrogel nerve conduit material is a conductive surface, and purple conductive nano polyaniline grows on the conductive surface along chitin fibers. The material prepared by the invention has good biocompatibility, antibacterial property and degradable property, and has no hemolysis.

Description

Preparation method of chitin hydrogel nerve conduit material capable of being sewn and cut into single-sided conductive and antibacterial material

Technical Field

The invention belongs to the field of polymer tissue engineering/medical polymer materials, and particularly relates to a preparation method of a single-side conductive antibacterial nano polyaniline chitin nerve conduit material.

Background

The high molecular base artificial tissue engineering nerve conduit bracket material becomes an important research field for repairing the nerve defect. The scaffold material is converted from non-degradable materials such as early silica gel and polytetrafluoroethylene to degradable materials such as collagen, chitin, chitosan, silk fibroin and polylactic acid. The existing nerve conduit material has the following defects: collagen has the defects of weak flexibility and strength, easy breakage, complex silk fibroin textile process and side effect of acidic degradation products of polylactic acid. Chitosan, a deacetylated product of chitin, is one of the most studied nerve conduit scaffold materials, and can be gelled by neutralization with alkali after being simply dissolved in an acidic aqueous solution, but the chitosan scaffold material is weak and fragile in strength, cannot be sewn, is usually poured to form a pipe, and is discontinuous in process and low in production efficiency. The chitosan catheter strength is reported to be improved by adding chitin powder or fibers or acylation into chitosan, but the improvement of the strength is limited by the limitation of a base material, but no nerve catheter prepared by direct chitin is reported. Although chitin has more advantages than chitosan, such as higher mechanical strength, coexistence of natural and protein collagen living tissues, good biocompatibility, longer degradation time than chitosan is beneficial to supporting effect on nerves, and inflammatory response is lower than that of chitosan. However, because the solubility of natural chitin is difficult and the natural chitin is not easy to process, most of the chitin nerve conduit materials reported in the prior art are substantially chitosan conduit materials or modified chitosan materials.

The function of single polymer conduit material for promoting nerve tissue regeneration is limited, and in recent years, researches show that composite conductive polymer polyaniline, polypyrrole, polythiophene and the like have a remarkable effect of promoting the regeneration of motor and sensory neuron axons, and the rigid powder cannot be self-molded and is usually used in combination with other polymer base materials. However, the hydrophobic large-particle conductor has the defect that residues in vivo are difficult to degrade, and long-term implantation can cause long-term delayed inflammation. Some reports have improved methods: the nano polyaniline or polypyrrole is prepared in advance and then is blended with the chitosan, but the nano conductor is unevenly distributed in the base material, has weak acting force with the matrix, is easy to separate and fall off, has poor conductivity, and often needs a large amount of nano conductor to form a continuous conductive path to form biological metabolism burden.

A novel nerve regeneration scaffold material which has high substrate strength, controllable substrate degradation speed, less inflammatory reaction, continuous conductivity of a nano conductive material, less consumption of a conductive body, stable combination with a substrate, regeneration promotion and metabolic degradation of a conductor is needed.

Disclosure of Invention

The invention provides a preparation method of a suturable single-side continuous conductive chitin hydrogel nerve conduit material, aiming at solving the problems of poor conductivity, discontinuous electric conductor, undegradability of the electric conductor, weak base material strength, inflammation of base material degradation products and the like of the existing degradable nerve conduit stent material.

The preparation operation steps of the suture-cutting single-side conductive antibacterial chitin hydrogel nerve conduit material are as follows:

(1) preparation of chitin hydrogel

Adding 1-9 g of pure chitin powder into 99-91 g of an alkali urea aqueous solution, wherein the alkali urea aqueous solution is an aqueous solution containing 10-16 wt% of potassium hydroxide and 4-8 wt% of urea, freezing at-30 to-40 ℃ for 1-3 h, stirring at room temperature, and thawing; alternately repeating the freezing-unfreezing operation for three times until the chitin is completely dissolved, and performing centrifugal defoaming to obtain bubble-free colorless transparent chitin solution; laying the chitin solution on a dry and smooth glass plate mold, standing for 1-12 h in a low-temperature coagulation bath, wherein the low-temperature coagulation bath is formed by slowly completing gelation at-5 ℃ below zero in an N, N dimethylacetamide aqueous solution with the concentration of 40-70wt% or ethanol with the concentration of 75-95 wt%; taking out the gel film, and washing with water for more than three times until no salt ions exist, thereby obtaining a colorless, soft, smooth and highly transparent chitin network hydrogel film;

(2) preparation of single-side continuous conductive antibacterial chitin hydrogel nerve conduit material

Cutting the chitin network hydrogel film into squares, clamping the squares in the middle of a U-shaped reactor, filling 150mL of conductive polymer monomer organic solution into one side pipe of the U-shaped reactor, and filling equal liquid level initiator phytic acid aqueous solution into the other side pipe of the U-shaped reactor; reacting for 1-4h in an ice bath condition, taking out the composite hydrogel membrane, and sequentially washing for 3 times by using ethanol and deionized water to obtain a single-sided conductive chitin hydrogel nerve conduit material; the structure is characterized in that one side surface of the chitin hydrogel is colorless, soft, smooth and highly transparent, the other side surface is a conductive antibacterial surface, and purple conductive nano polyaniline grows along chitin fibers on the conductive antibacterial surface;

cutting the single-side conductive antibacterial chitin hydrogel nerve conduit material into a rectangle with a required size, facing the conductive surface antibacterial surface inwards, and sewing with an operation thread to form a tubular nerve conduit; the mechanical strength of the single-side conductive antibacterial chitin hydrogel nerve conduit material is more than 10 times of that of the traditional chitosan conduit material.

The further technical scheme is as follows:

in the step (1), the pure chitin powder is alpha chitin powder, beta chitin powder, or a mixed powder of alpha chitin powder and beta chitin powder.

The alpha chitin powder is shrimp alpha chitin powder, crab shell alpha chitin powder and mixed alpha chitin powder; the mixed alpha chitin powder is prepared from the following components in a mass ratio of 2: 1. 1: 1. 1: 2 is prepared by evenly mixing the shrimp shell alpha chitin powder and the crab shell alpha chitin powder.

The beta chitin powder is squid bone beta chitin powder, and the mixed chitin powder is prepared from the following components in a mass ratio of 1: 1 is prepared by evenly mixing dried small shrimp alpha chitin powder or crab shell alpha chitin powder with squid bone beta chitin powder.

In the step (1), the mass concentration of chitin in the chitin solution is as follows: 3-9% of pure alpha chitin, 1-5% of pure squid bone beta chitin and 3-7% of mixed chitin.

In the step (1), the length and width of the chitin network hydrogel film are not limited and are controlled by the size of a mould, but the thickness is controlled to be 0.15-0.5 mm.

In the step (1), the organic solution of the conductive polymer monomer is a toluene solution of aniline monomer with the concentration of 0.1 mol/L.

In the step (1), the initiator phytic acid aqueous solution is an aqueous solution containing 0.025 mol/L-0.15 mol/L ammonium persulfate and 5wt% -20 wt% phytic acid bridging agent.

The beneficial technical effects of the invention are embodied in the following aspects:

1. the chitin nano-fiber network hydrogel with high strength and high transparency is obtained by adopting a green solvent and a controllable solidification technology as a nerve conduit material base material, the mechanical strength is about more than 10 times of that of a traditional chitosan conduit, the degradation period is adjustable by molecular weight, and the pipe diameter can be freely sewed and adjusted to accurately match the requirements of different defect parts.

2. Under the triple combined action of an interface effect, low-temperature growth control and nanofiber network confinement, ultra-small nano polyaniline which grows on one side by virtue of a phytic acid bridge is obtained and used as the inner wall of the catheter, the dosage of a conductor only adopts 1/10-1/20 of the traditional mixing method to realize continuous conduction, the polyaniline has a small molecular weight (1-2 ten thousand), can be quickly phagocytized by phagocytes to realize biological metabolism, does not need secondary operation to remove, and meanwhile, the material has excellent antibacterial property under the double mechanisms of an electroactive mechanism and phytic acid charge attraction, and is beneficial to bacteriostasis and inflammation diminishing during tissue regeneration.

Drawings

FIG. 1 is a diagram of a reaction apparatus in step (2).

Fig. 2 is a photograph of the appearance of the single-sided conductive chitin hydrogel obtained in example 2.

Fig. 3 is a photograph of the single-sided conductive chitin hydrogel of example 3 observed under a fluorescence microscope, showing that one side is transparent and one side is dark green, and the single-sided conductive chitin hydrogel is composed of nano polyaniline.

Fig. 4 is a graph showing that the principle of stable loading of the conductive polyaniline nanoparticles on the chitin nanofibers is due to the bridging effect of phytic acid.

Fig. 5 is a photograph of the LED bulb of example 6, which can be lit by connecting the single-sided conductive chitin hydrogel to the LED bulb.

Fig. 6a is a SEM image of the shell surface of pure crab shell of the single-sided conductive chitin hydrogel nerve conduit material of example 4.

Fig. 6b is a SEM image of the grown nano-polyaniline of the single-sided conductive chitin hydrogel nerve conduit material of example 4; the hydrogel is shown to be a nano-thorn ball type hydrogel which takes a chitin nano-fiber network as a framework and polyaniline only grows on a single surface.

FIG. 7a is a photograph of an observation of hemolysis experiment in Triton X-100 on a nerve conduit material of example 3, example 4, example 7 in a PBS solution of rabbit red blood cells; shows that pure chitin and different single-side conductive chitin hydrogel catheter materials have no hemolysis.

FIG. 7b is a microscopic SEM photograph of rabbit red blood cells in PBS solution after hemolysis experiment.

FIG. 7c is a microscopic SEM photograph of rabbit red blood cells in a hemolysis experiment on the material of example 7.

FIG. 7d is a microscopic SEM photograph of rabbit red blood cells in a hemolysis experiment on the material of example 3.

FIG. 7e is a microscopic SEM photograph of rabbit red blood cells in a hemolysis experiment on the material of example 4.

FIG. 7f is a microscopic SEM photograph of rabbit red blood cells in Triton X-100.

FIG. 7g is a graph of recorded hemolysis rate data for example 3, example 4 and example 7; the data in fig. 7 show that the nano polyaniline chitin hydrogel material has no hemolysis.

FIG. 8a is an SEM image of cell growth observed by freeze-drying after culturing nerve cells on the material of example 4.

Fig. 8b is a partial enlarged view of fig. 8 a. The nano polyaniline chitin hydrogel material on the surface is very beneficial to the climbing growth of nerve cell tentacles.

FIG. 9 shows the results of the antimicrobial experiments on three types of infectious bacteria (E.coli, Staphylococcus aureus, Pseudomonas aeruginosa) commonly found in wards, where a is example 1, b is reference group example 7, c is example 4, d is example 3, and e is example 5. The pure chitin hydrogel has no obvious bacteriostatic zone, and the nano polyaniline/chitin hydrogel has obvious bacteriostatic zone, which is attributed to the double mechanisms of nano polyaniline electric activity and phytic acid bridging agent to endow the chitin hydrogel material with excellent antibacterial property.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

Example 1

The preparation operation steps of the single-side conductive nano polyaniline chitin nerve conduit stent material are as follows:

(1) preparation of dried small shrimp chitin hydrogel film

Weighing 4g of pure dried small shrimp alpha chitin powder, adding the powder into 100g of mixed solution, uniformly mixing the mixed solution with 11g of potassium hydroxide, 4g of urea and 85g of water, and freezing for 2 hours at-30 ℃; unfreezing at room temperature; alternately repeating the freezing and unfreezing operations for three times until the solution is completely dissolved; centrifuging and defoaming the solution at the rotating speed of 8000rpm for 12min to obtain a colorless and transparent shrimp bran alpha chitin solution; laying the dried small shrimp alpha chitin solution on a dry and smooth glass plate mould, immersing the dried small shrimp alpha chitin solution into an N, N dimethylacetamide aqueous solution with the mass concentration of 60 percent at the temperature of minus 5 ℃, standing and storing for 4 hours to slowly complete gelation; taking out the gel film, washing with water for several times until no salt ion exists, and obtaining a colorless, soft, smooth and highly transparent dried small shrimp alpha chitin network hydrogel film with the length of 40cm, the width of 10cm and the thickness of 0.25 mm.

(2) Preparation of single-side conductive nano polyaniline-dried small shrimp chitin nerve conduit material

Referring to an experimental device diagram shown in figure 1, a shrimp alpha chitin network hydrogel film is cut into a square of 8cm and clamped in the middle of a U-shaped reactor, 150mL of toluene solution of aniline monomer with the concentration of 0.1mol/L is added into a pipe orifice at one side of the U-shaped reactor, 150mL of aqueous solution containing 0.05mol/L ammonium persulfate and 5wt% of phytic acid is added into a pipe orifice at the other side of the U-shaped reactor, the system is placed in an ice bath condition for reaction for 2 hours, after the device is disassembled, the composite hydrogel film is taken out and washed with ethanol and deionized water for several times, and the single-side conductive antibacterial chitin hydrogel nerve conduit material is obtained, wherein one side of the nerve conduit material is still colorless, soft, smooth and highly transparent chitin hydrogel, and the other side of the nerve conduit material is a conductive surface which is formed by growing purple conductive nano polyaniline along chitin fibers.

The chitin hydrogel nerve conduit material with single-side conductivity prepared in the example 1 was cut into a rectangle with a desired size, the dark conductive side was turned inside, and the material was sutured into a tubular nerve conduit material with an operation thread. It is fully degradable, non-hemolytic, electrically conductive, and can stimulate nerve regeneration. The single-side conductive chitin hydrogel nerve conduit scaffold material prepared in the embodiment 1 has the mechanical tensile strength of 1.6MPa, the elongation at break of 67%, and the cell is easy to attach and grow during cell culture, has good biocompatibility and no hemolysis, and is shown in fig. 9, wherein a sample a in the embodiment has an obvious antibacterial ring (diameter of 9-11 mm) for three types of common infectious bacteria (escherichia coli, staphylococcus aureus and pseudomonas aeruginosa) in a ward, and shows excellent antibacterial property.

Example 2

The preparation operation steps of the single-side conductive antibacterial nano polyaniline chitin nerve conduit stent material are as follows:

(1) squid bone beta chitin hydrogel membrane

Weighing 3g of pure squid bone beta chitin powder, adding the powder into 100g of mixed solution, uniformly mixing the mixed solution with 16g of potassium hydroxide, 5g of urea and 79g of water, and freezing the mixed solution at the temperature of minus 35 ℃ for 1 hour; unfreezing; alternately repeating the freezing and unfreezing operations for three times until the solution is completely dissolved; centrifuging and defoaming the solution at the rotation speed of 8000rpm for 12min to obtain a colorless and transparent squid bone beta chitin solution, spreading the squid bone beta chitin solution on a dry and smooth glass plate mold, immersing the squid bone beta chitin solution in an ethanol aqueous solution with the mass concentration of 90wt% at the temperature of-5 ℃, standing and storing for 2h to slowly complete gelation; taking out the gel film, washing with water for several times until no salt ion exists, obtaining a colorless, soft, smooth and highly transparent squid bone beta chitin network hydrogel film with the length of 30cm, the width of 15cm and the thickness of 0.2mm, and refrigerating and storing in ethanol with the mass concentration of 80 wt%.

(2) Preparation of single-side conductive antibacterial chitin hydrogel nerve conduit stent material

Cutting the squid bone beta chitin network hydrogel membrane into 8cm squares, clamping the squares in the middle of a U-shaped reactor, adding 150mL of toluene solution of aniline monomer with the concentration of 0.1mol/L into a pipe orifice at one side of the U-shaped reactor, adding 150mL of aqueous solution containing 0.025mol/L ammonium persulfate and 10% phytic acid into a pipe orifice at the other side of the U-shaped reactor, placing the system in an ice bath condition for reaction for 1h, removing the device, taking out the composite hydrogel membrane, washing the composite hydrogel membrane with ethanol and deionized water for several times to obtain a single-sided conductive chitin hydrogel nerve conduit support material, wherein the size of the single-sided conductive chitin hydrogel nerve conduit support material is the same as that in the step (1); one side surface of the single-side conductive chitin hydrogel nerve conduit support material is still colorless, soft, smooth and highly transparent beta chitin hydrogel, and the other side surface is grown with green conductive nano polyaniline along chitin fibers, as shown in a sample appearance picture of figure 2.

The single-sided conductive antibacterial chitin hydrogel nerve conduit stent material prepared in the embodiment 2 is cut into a rectangle with a required size, the dark conductive side faces inwards, and the dark conductive side faces are sewn into a tubular nerve conduit material by using an operation thread. It is fully degradable, has no hemolytic property, can conduct electricity, and can promote nerve tissue regeneration. The single-sided conductive antibacterial chitin hydrogel nerve conduit scaffold material prepared in the embodiment 2 has the mechanical tensile strength of 2.2 MPa and the elongation at break of 75%.

Example 3

The preparation operation steps of the single-side conductive nano antibacterial polyaniline chitin nerve conduit stent material are as follows:

(1) preparation of crab shell chitin hydrogel film

Weighing 7g of pure crab shell alpha chitin powder, adding the powder into 99g of mixed solution, uniformly mixing the mixed solution with 10g of potassium hydroxide, 4g of urea and 85g of water, and freezing for 1h at-30 ℃; mechanically stirring and unfreezing; freezing, thawing and stirring are alternately repeated for three times until the crab shells are completely dissolved to obtain a crab shell alpha chitin solution; spreading the crab shell alpha chitin solution on a dry and smooth glass plate mold, soaking the crab shell alpha chitin solution in 95% ethanol at the temperature of 5 ℃, standing and storing for 6 hours to slowly complete gelation; taking out the gel film, washing with water for several times until no salt ion exists, obtaining a colorless, soft, smooth and highly transparent crab shell alpha chitin network hydrogel film with the length of 40cm, the width of 20cm and the thickness of 0.4mm, and storing in 20% ethanol.

Cutting the crab shell alpha chitin network hydrogel membrane into 8cm squares, clamping the square squares in the middle of a U-shaped reactor, adding 150mL of aniline toluene solution with the concentration of 0.1mol/L into a pipe orifice at one side of the U-shaped reactor, adding an aqueous solution containing 0.1mol/L ammonium persulfate and phytic acid with the concentration of 5wt% at the same liquid level into a pipe orifice at the other side of the U-shaped reactor, placing the system in an ice bath condition for reaction for 1h, dismantling the device, taking out the composite hydrogel membrane, washing the composite hydrogel membrane with ethanol and deionized water for a plurality of times to obtain the single-sided conductive chitin hydrogel nerve conduit support material, wherein the size of the single-sided conductive chitin hydrogel nerve conduit support material is the same as that in the step (1), one side surface of the single-sided conductive chitin hydrogel nerve conduit support material is still colorless, soft, smooth and highly transparent alpha chitin hydrogel, and the other side surface of the single-sided conductive chitosan hydrogel nerve conduit support material grows deep purple nano polyaniline along chitin fibers, and the growth of the polyaniline is observed to present gradient growth distribution under a fluorescence microscope as shown in a figure 3, gradually shallowing it to one side without polyaniline. .

Referring to fig. 7a, a photograph of an experimental observation of hemolysis of rabbit red blood cells on the nerve conduit material of example 3 in PBS and in Triton X-100 shows no hemolysis. Referring to fig. 7d, microscopic SEM image of hemolysis experiment of rabbit red blood cells on the material of example 3, and fig. 7g, recorded plot of hemolysis rate data of example 3; the material of example 3 is shown to be non-hemolytic. Referring to FIG. 9, sample d of this example has an obvious antibacterial ring (diameter 15-17 mm) against three types of infectious bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa) common in sickrooms, and shows excellent antibacterial property.

The single-sided conductive antibacterial chitin hydrogel nerve conduit stent material prepared in the embodiment 3 is cut into a rectangle with a required size, the dark conductive side faces inwards, and the dark conductive side faces are sewn into a tubular nerve conduit material by using an operation thread. It is fully degradable, non-hemolytic, electrically conductive, and can stimulate nerve regeneration. The single-sided conductive antibacterial chitin hydrogel nerve conduit scaffold material prepared in the embodiment 3 has the mechanical tensile strength of 1.3 MPa and the elongation at break of 62%.

Example 4

(1) preparation of crab shell chitin hydrogel film

Weighing 7g of pure crab shell alpha chitin, adding the pure crab shell alpha chitin into 100g of mixed solution, uniformly mixing the mixed solution with 11g of potassium hydroxide, 4g of urea and 85g of water, and freezing for 1h at the temperature of minus 30 ℃; mechanically stirring until completely dissolving to obtain crab shell alpha chitin solution, spreading the crab shell alpha chitin solution on a dry and smooth glass plate mold, soaking the crab shell alpha chitin solution in 95% ethanol with the concentration of 5 ℃, standing and storing for 6 hours to slowly complete gelation, taking out a gel film, washing for several times until no salt ions exist to obtain a colorless, soft, smooth and highly transparent crab shell alpha chitin network gel film with the length of 40cm, the width of 20cm and the thickness of 0.4mm, and refrigerating and storing in 20% ethanol.

Cutting the crab shell alpha chitin network hydrogel film into 8cm squares, clamping the crab shell alpha chitin network hydrogel film in the middle of a U-shaped reactor, adding 150mL of toluene solution of aniline monomer with the concentration of 0.1mol/L into a pipe orifice at one side of the U-shaped reactor, adding 150mL of aqueous solution containing 0.1mol/L ammonium persulfate and phytic acid with the concentration of 10% into a pipe orifice at the other side of the U-shaped reactor, placing the system in an ice bath condition for reaction for 3h, dismantling the device, taking out the composite hydrogel film, washing the composite hydrogel film with ethanol and deionized water for several times to obtain the single-side conductive chitin hydrogel nerve conduit material, wherein the size of the nerve conduit material is the same as that of the step (1), one side surface of the nerve conduit material is colorless, soft, smooth and highly transparent alpha chitin hydrogel, and the other side surface of the nerve conduit material grows deep purple conductive nano polyaniline along chitin fibers.

Referring to FIG. 7a, a photograph of a hemolysis experiment observation of rabbit red blood cells on the nerve conduit material of example 4 in PBS and in Triton X-100; the material of example 4 is shown to be non-hemolytic. Referring to fig. 7e, microscopic SEM image of rabbit red blood cells in hemolysis experiment on the material of example 4; referring to fig. 7g, a plot of hemolysis rate data for example 4 is recorded; the data in FIG. 7 above show that the material of example 4 is not hemolytic.

Referring to fig. 6a, SEM of the shell surface of pure crab shell of the single-sided conductive chitin hydrogel nerve conduit material of example 4, and SEM of the nano-polyaniline grown on the other side (see fig. 6 b), show that the nano-polyaniline is in a spiky ball-shaped state. Referring to fig. 8a and 8b, SEM images of cell growth observed by freeze-drying after culturing nerve cells on the material of this example 4 show that the surface nano polyaniline chitin hydrogel material is very beneficial for the tentacle growth of nerve cells.

In addition, referring to fig. 9, sample c in this example 4 has a significant antibacterial circle (diameter 18-20 mm) against three types of infectious bacteria (escherichia coli, staphylococcus aureus, pseudomonas aeruginosa) common in sickrooms, and shows excellent antibacterial property.

The single-sided conductive antibacterial chitin hydrogel nerve conduit stent material of the embodiment 4 is cut into a rectangle with a required size, the dark conductive side faces inwards, and the catheter is sewn into a tubular nerve conduit material by an operation thread. It is fully degradable, non-hemolytic, electrically conductive, and can stimulate nerve regeneration. The single-sided conductive antibacterial chitin hydrogel nerve conduit scaffold material prepared in the embodiment 4 has the mechanical tensile strength of 1.25 MPa and the elongation at break of 56%.

Example 5

The preparation operation steps of the single-side conductive antibacterial nano polyaniline chitin nerve conduit material are as follows:

(1) preparation of shrimp shell crab shell chitin hydrogel film

Weighing 3g of pure shrimp shell alpha chitin powder and 3g of pure crab shell alpha chitin powder, adding into 100g of mixed solution, uniformly mixing the mixed solution with 11g of potassium hydroxide, 5g of urea and 84g of water, and freezing for 3h at-30 ℃; unfreezing and mechanically stirring; freezing, thawing and stirring are alternately repeated for three times until the mixture is completely dissolved; centrifuging and defoaming the solution at the rotating speed of 9000rpm for 10min to obtain a colorless transparent alpha chitin solution from the shrimp and crab shells; spreading the alpha chitin solution on a dry and smooth glass plate mold, soaking the mold in 90% ethanol at 0 ℃, standing and storing for 2 hours to slowly complete gelation; taking out the gel film, washing with water for several times until no salt ion exists, obtaining a colorless, moderate-softness, smooth and highly transparent crayfish shell alpha chitin network hydrogel film with the length of 15cm, the width of 10cm and the thickness of 0.25mm, and storing in ethanol with the concentration of 70%.

(2) Preparation of single-side conductive antibacterial chitin hydrogel nerve conduit material

Cutting the alpha chitin network hydrogel film of the crab shell of the dried small shrimps into a square of 8cm, clamping the square in the middle of a U-shaped reactor, adding 150mL of a toluene solution of an aniline monomer with the concentration of 0.1mol/L into a pipe orifice at one side of the U-shaped reactor, adding 150mL of an ammonium persulfate with the concentration of 0.05mol/L and a phytic acid solution with the concentration of 20% into a pipe orifice at the other side of the U-shaped reactor, placing the system in an ice bath condition for reaction for 3h, dismantling the device, taking out the composite hydrogel film, washing with ethanol and deionized water for several times to obtain the single-side conductive antibacterial chitin hydrogel nerve conduit support material, wherein one side of the single-side conductive antibacterial chitin hydrogel nerve conduit support material is colorless, soft, smooth and highly transparent alpha chitin hydrogel, and the other side of the single-side of the conductive chitin nerve conduit support material is dark-colored by growing dark purple nano polyaniline capable of being conductive along chitin fibers.

The single-sided conductive antibacterial chitin hydrogel nerve conduit stent material of the embodiment 5 is cut into a rectangle with a required size, the dark conductive side faces inwards, and the catheter is sewn into a tubular nerve conduit material by an operation thread. It is fully degradable, non-hemolytic, electrically conductive, and can stimulate nerve regeneration. The single-sided conductive antibacterial chitin hydrogel nerve conduit scaffold material prepared in the embodiment 5 has the mechanical tensile strength of 1.4 MPa and the elongation at break of 54%. In addition, referring to fig. 9, sample e in this example has an obvious antibacterial circle (diameter of 16-17 mm) against three types of common infectious bacteria (escherichia coli, staphylococcus aureus, pseudomonas aeruginosa) in ward, and shows excellent antibacterial property.

Example 6

(1) preparation of crab shell-squid bone mixed chitin network hydrogel film

Weighing 2g of pure squid bone beta chitin powder and 2g of pure crab shell alpha chitin powder, adding into 100g of mixed solution, uniformly mixing the mixed solution with 15g of potassium hydroxide, 4g of urea and 81g of water, and freezing for 3 hours at-40 ℃; mechanically stirring and unfreezing; alternately repeating the freezing and unfreezing operations for three times until the solution is completely dissolved; centrifuging at 9000rpm for 10min to obtain colorless and transparent crab shell-squid bone mixed chitin solution, spreading the crab shell-squid bone mixed chitin solution on a dry and smooth glass plate mold, soaking in 85% ethanol at 0 deg.C, standing for 3h for slow gelation, taking out the gel film, washing with water for several times until no salt ion is present to obtain a colorless, soft, smooth and highly transparent crab shell-squid bone mixed chitin network hydrogel film with length of 35cm, width of 25cm square and thickness of 0.35mm, and refrigerating in 70% ethanol for preservation.

Cutting the crab shell and squid bone mixed chitin network hydrogel film into 8cm squares, clamping the square squares in the middle of a U-shaped reactor, adding 150mL of toluene solution of aniline monomer with the concentration of 0.1mol/L into a pipe orifice at one side of the U-shaped reactor, adding 150mL of aqueous solution containing ammonium persulfate with the concentration of 0.0125mol/L and phytic acid with the concentration of 15% into a pipe orifice at the other side of the U-shaped reactor, placing the system in an ice bath condition for reaction for 3 hours, dismantling the device, taking out the composite hydrogel film, washing with ethanol and deionized water for several times, and obtaining the single-side conductive chitin hydrogel nerve conduit material, wherein one side of the nerve conduit material is colorless, soft, smooth and highly transparent crab shell and squid bone mixed chitin hydrogel, and the other side of the nerve conduit material is grown with dark green conductive nano polyaniline along chitin fibers.

Referring to fig. 5, the single-sided conductive chitin hydrogel is connected with the LED bulb and then the bulb can be lighted by electrifying.

The single-sided conductive antibacterial chitin hydrogel nerve conduit material of the embodiment 6 is cut into a rectangle with a required size, the dark conductive side faces inwards, and the nerve conduit material is sewn into a tubular nerve conduit material by an operation thread. It is fully degradable, non-hemolytic, electrically conductive, and can stimulate nerve regeneration. The single-sided conductive antibacterial chitin hydrogel nerve conduit material prepared in the embodiment 6 has the mechanical tensile strength of 1.8 MPa and the elongation at break of 63%.

Example 7

This example provides a pure chitin catheter material as a reference group, and the preparation steps of the method are as follows:

weighing 5g of crab shell chitin alpha chitin powder, adding the crab shell chitin alpha chitin powder into a solution consisting of 11g of potassium hydroxide, 4g of urea and 85g of water, freezing for 2 hours at the temperature of minus 30 ℃, unfreezing and mechanically stirring, and repeatedly freezing and unfreezing for three times until the crab shell chitin alpha chitin powder is completely dissolved. Centrifuging and defoaming the solution at the rotating speed of 9000rpm for 10min to obtain a colorless transparent alpha chitin solution, laying the solution on a glass plate mold, immersing the solution in 90% ethanol at the temperature of 0 ℃, standing and storing for 3h to slowly complete sol gelation, taking out the solution, washing the solution for several times until no salt ions exist to obtain a piece of colorless, soft, smooth and highly transparent crab shell alpha chitin network hydrogel film with the length of 35cm, the width of 25cm square and the thickness of 0.35mm, storing the crab shell alpha chitin network hydrogel film in deionized water, cutting the crab shell alpha chitin network hydrogel film into a rectangle with required size, and sewing the crab shell alpha chitin network hydrogel film into a tube by using an operation, namely the pure alpha chitin hydrogel nerve conduit material is used as a reference group. It is colorless transparent hydrogel, non-toxic, degradable, but not conductive. The mechanical tensile strength of the pure α chitin hydrogel nerve conduit material of this example 7 was 0.8MPa, and the elongation at break was 53%.

Referring to fig. 7a, a photograph of an experimental observation of hemolysis of rabbit red blood cells on the nerve conduit material of example 7 in PBS and in Triton X-100 shows no hemolysis. Referring to fig. 7c, microscopic SEM image of rabbit red blood cells in hemolysis experiment on the material of example 7; referring to fig. 7g, a plot of the hemolysis rate data for example 7 is recorded; the data in fig. 7 above show that the pure chitin hydrogel material is not hemolytic. Referring to fig. 9, the sample b in this example has no obvious visible antibacterial zone for three types of common infectious bacteria (escherichia coli, staphylococcus aureus, pseudomonas aeruginosa) in a ward, and the antibacterial effect of the pure chitin hydrogel material is not obvious.

It will be understood by those skilled in the art that the foregoing is merely a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included within the scope of the present invention.

Claims

1. A preparation method of a chitin hydrogel catheter material capable of being sewn and cut into a single-side conductive and antibacterial material is characterized by comprising the following operation steps:

(1) preparation of chitin hydrogel

Adding 1-9 g of pure chitin powder into 99-91 g of an alkali urea aqueous solution, wherein the alkali urea aqueous solution is an aqueous solution containing 10-16 wt% of potassium hydroxide and 4-8 wt% of urea, freezing at-30 to-40 ℃ for 1-3 h, stirring at room temperature, and thawing; freezing-unfreezing alternate repeated operation is carried out for three times until the chitin is completely dissolved, and centrifugal deaeration is carried out to obtain bubble-free colorless transparent chitin solution; laying the chitin solution on a dry and smooth glass plate mold, standing for 1-12 h in a low-temperature coagulation bath, wherein the low-temperature coagulation bath is formed by slowly finishing gelation at-5 ℃ below zero in 40-70wt% N, N dimethylacetamide aqueous solution or 75-95 wt% ethanol; taking out the gel film, and washing with water for more than three times until no salt ions exist, thereby obtaining a colorless, soft, smooth and highly transparent chitin network hydrogel film;

Cutting the chitin network hydrogel film into squares, clamping the squares in the middle of a U-shaped reactor, filling 150mL of conductive polymer monomer organic solution into one side pipe of the U-shaped reactor, and filling equal liquid level initiator phytic acid aqueous solution into the other side pipe of the U-shaped reactor; reacting for 1-4h in an ice bath condition, taking out the composite hydrogel membrane, and sequentially washing for 3 times by using ethanol and deionized water to obtain a single-sided conductive chitin hydrogel nerve conduit material; the structure is characterized in that one side surface is colorless, soft, smooth and highly transparent chitin hydrogel, the other side surface is a conductive surface, and purple conductive nano polyaniline grows along chitin fibers on the conductive surface;

cutting the single-sided conductive antibacterial chitin hydrogel nerve conduit material into a rectangle with a required size, facing the conductive surface inwards, and sewing with an operation thread to form a tubular nerve conduit; the mechanical strength of the single-side conductive chitin hydrogel nerve conduit material is more than 10 times of that of the traditional chitosan conduit material.

2. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the pure chitin powder is alpha chitin powder, beta chitin powder, or a mixed powder of alpha chitin powder and beta chitin powder.

3. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 2, wherein the method comprises the following steps: the alpha chitin powder is shrimp alpha chitin powder, crab shell alpha chitin powder and mixed alpha chitin powder; the mixed alpha chitin powder is prepared from the following components in a mass ratio of 2: 1. 1: 1. 1: 2 is prepared by evenly mixing the shrimp shell alpha chitin powder and the crab shell alpha chitin powder.

4. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 2, wherein the method comprises the following steps: the beta chitin powder is squid bone beta chitin powder, and the mixed chitin powder is prepared from the following components in a mass ratio of 2: 1. 1: 1. 1: 2 is prepared by evenly mixing dried small shrimp alpha chitin powder or crab shell alpha chitin powder with squid bone beta chitin powder.

5. The method for preparing the suture-cuttable single-sided conductive antibacterial chitin hydrogel nerve conduit material according to claim 2, which is characterized in that: in the step (1), the mass concentration of chitin in the chitin solution is as follows: 3-9% of pure alpha chitin, 1-5% of pure squid bone beta chitin and 3-7% of mixed chitin.

6. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the length and width of the chitin network hydrogel film are not limited, but the thickness is controlled to be 0.15-0.75 mm.

7. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the organic solution of the conductive polymer monomer is a toluene solution of aniline monomer with the concentration of 0.1 mol/L.

8. The preparation method of the suturable single-sided conductive antibacterial chitin hydrogel nerve conduit material as claimed in claim 1, wherein the method comprises the following steps: in the step (1), the initiator phytic acid aqueous solution is an aqueous solution containing ammonium persulfate with the concentration of 0.025 mol/L-0.15 mol/L and phytic acid bridging agent with the concentration of 5wt% -20 wt%.