CN114533967B - Chitosan-containing nerve conduit and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a chitosan nerve conduit and a preparation method thereof. The chitosan nerve conduit is divided into a short-term conduit, a medium-term conduit and a long-term conduit; wherein the short-term catheter degradation time is 7-30 days, the porosity is 75-85%, and the deacetylation degree of the raw material chitosan: DD is between 70% and 75%; the medium-term catheter degradation time is 31-90 days, the porosity is 70-75%, and the deacetylation degree of the raw material chitosan: DD is more than 75 percent and less than or equal to 90 percent; the degradation time of the long-term catheter is 91-200 days, the porosity is 65-70%, and the deacetylation degree of the raw material chitosan is more than 90% and less than or equal to DD and 98%. The invention makes the degradation time of the nerve conduit more accord with the nerve repair growth rule by controlling the porosity of the nerve conduit and the deacetylation degree of the chitosan raw material, and can formulate the nerve conduit with corresponding repair time according to the nerves at different parts, different defect lengths and the thickness of defective nerves.

Description

Chitosan-containing nerve conduit and preparation method thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a chitosan nerve conduit and a preparation method thereof.

Background

Repair of peripheral nerve injury is a challenging medical problem, autologous nerve transplantation is the "gold standard" in peripheral nerve repair due to its non-immunogenic role, but there are also problems with limited supply of donor tissue, risk of neuroma formation, size mismatch and axonal distribution between the donor nerve and the injury site. The nerve conduit is a tubular device made of natural or/and synthetic polymer materials, the nerve segment end is placed into the tube to be sutured, and the nerve axon can grow along the tube cavity. The nerve conduit can provide a physical barrier function for the invasion of connective tissues to the nerve segment end, and scar formation is prevented; is favorable for the accumulation of neurotrophic factors, guides the axon axial growth of neurons, avoids the formation of neuroma, and utilizes the chemotaxis of distal nerves to repair peripheral nerve defects with longer distance. However, poor biocompatibility and low bioactivity are major drawbacks of most artificial nerve conduits.

Chitosan (CS) is a natural biological material, is the only known basic polysaccharide with positive charge at present, has good biocompatibility, biodegradability and antibacterial property, can support the adhesion and growth of Schwann cells, can inhibit the growth of fibrocytes and the formation of scars, and promotes the growth of endothelial cells, thus being an ideal nerve conduit material. For example, chinese patent 01108208.9 discloses a medical artificial nerve graft, which consists of a catheter and a fibrous scaffold, wherein the catheter consists of chitosan, polyglycolic acid and polylactic acid, and the fibrous scaffold is made of polyglycolic acid or polylactic acid. For another example, chinese patent 02113103.1 discloses a chitosan medical nerve, which is composed of a catheter and a fibrous scaffold, wherein the catheter is composed of chitosan, and the fibrous scaffold is made of chitosan. The above patents all use chitosan as raw material to prepare nerve repair graft, and although the problem of poor general compatibility and bioactivity of nerve conduit is solved, the degradation time of the prepared graft is uncontrollable, and the graft can not perfectly match with nerve defects of different parts, different lengths and different thicknesses, resulting in poor repair effect or easy occurrence of adverse reaction.

Disclosure of Invention

Based on the defects of the prior art, the invention provides the chitosan nerve conduit capable of accurately controlling the degradation time and the preparation method thereof.

Therefore, the invention provides a chitosan nerve conduit which is divided into a short-term conduit, a medium-term conduit and a long-term conduit; wherein the short-term degradation time of the catheter is 7-30 days, the porosity is 75-85%, and the Deacetylation Degree (DD) of the chitosan is as follows: DD is between 70% and 75%; the medium-term catheter degradation time is 31-90 days, the porosity is 70-75%, and the deacetylation degree of chitosan is as follows: DD is more than 75 percent and less than or equal to 90 percent; the degradation time of the long-term catheter is 91-200 days, the porosity is 65-70%, and the deacetylation degree of the raw material chitosan is more than 90% and less than or equal to DD and 98%.

The invention further provides a preparation method of the chitosan nerve conduit, which comprises the following steps:

(1) Dissolving chitosan, separating by 1-10 ion exchange columns with diameter of 2-5cm and length of 20-100cm, eluting, treating with alkaline solution, and drying to obtain chitosan;

(2) Dissolving chitosan, injecting, freezing, devitrifying, solidifying and forming, treating with alkaline solution, and freeze-drying to obtain the nerve conduit.

Preferably, in order to omit the artificial suture of the nerve conduit, the present invention further comprises a step of mixing chitosan with acidic amino acid and dopamine before the injection molding step of step (2). Carboxyl in the acidic amino acid can simultaneously form amido bond with chitosan and amino in human nerves, thereby realizing the self-connection of the nerve conduit and the nerves; meanwhile, dopamine in an alkaline solution can form polydopamine with high biological adhesion, and the polydopamine and an amido bond together improve the adhesion between the nerve conduit and the nerve, so that the step of manual suturing is omitted.

Preferably, the mass ratio of the chitosan to the acidic amino acid to the dopamine is 100.

Preferably, the step of mixing chitosan with acidic amino acid and dopamine further comprises a step of activating the acidic amino acid by a catalyst.

Preferably, the mass ratio of the catalyst to the acidic amino acid is 10.

Preferably, the catalyst is selected from one or two of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

Preferably, the acidic amino acid is selected from one or two of glutamic acid and aspartic acid.

Preferably, in the step (2), the devitrification temperature is-10 ℃ to-30 ℃ and the time is 1-10h.

Preferably, in the step (2), the forming method is freeze drying, the temperature is-20 ℃ to-100 ℃, the vacuum degree is 0-200Pa, and the time is 12-24h.

Preferably, the ion exchange column in step (1) is one or more selected from a sephadex column and a sepharose column.

Preferably, the eluent in the step (1) is acetic acid-sodium acetate buffer solution containing 0.1-2mol/L NaCl.

Preferably, the deacetylation degree of the chitosan obtained in the step (1) is 70-98%. According to the different charges of chitosan with different deacetylation degrees in the solution, chitosan solutions with deacetylation degrees of 70-98% can be respectively separated from the 1 st-10 th ion exchange columns.

Preferably, the alkaline solution in step (1) and/or step (2) is selected from one or two of NaOH and KOH solution.

Preferably, the mass percentage concentration of the alkaline solution is 10-80%.

The invention has the beneficial effects that:

1. the invention can make nerve conduits with corresponding repair time according to the nerves of different parts, different defect lengths and the thickness of defective nerves: wherein the short-term catheter degradation time is 7-30 days, the porosity is 75-85%, and the deacetylation degree of the raw material chitosan: DD is between 70% and 75%; the medium-term catheter degradation time is 31-90 days, the porosity is 70-75%, and the deacetylation degree of the raw material chitosan: DD is more than 75 percent and less than or equal to 90 percent; the degradation time of the long-term catheter is 91-200 days, the porosity is 65-70%, and the deacetylation degree of the raw material chitosan is more than 90% and less than or equal to DD and 98%. The porosity of the nerve conduit and the deacetylation degree of the chitosan serving as the raw material are controlled, so that the degradation time of the nerve conduit is more consistent with the nerve repair growth rule, the side effects of over-fast degradation and incomplete recovery of nerves or over-slow degradation causing inflammation and the like are avoided, the whole using effect is good, and the clinical popularization is facilitated.

Chitosan is degraded by the influence of lysozyme in vivo into mono-or oligosaccharides of glucosamine and acetylglucosamine. Chitosan is a natural high molecular biological material directly prepared from animals, is influenced by animal species, growth conditions and individual differences, and if the non-screened chitosan is directly used for preparing the catheter, the degradation time of the catheter cannot be accurately controlled in the degradation process of the catheter. The deacetylation degree of chitosan is related to the action efficiency of chitosan with lysozyme in the degradation process, the higher the deacetylation degree of chitosan is, the longer the degradation time is, and the lower the deacetylation degree is, the shorter the degradation time is.

2. According to the invention, an ion exchange chromatography technology is introduced in chitosan deacetylation degree screening, and according to different charges carried by chitosan molecules with different deacetylation degrees in a solution (the higher the deacetylation degree is, the more the charges are carried by the chitosan molecules), the solution sequentially passes through 1-10 ion exchange columns with the diameter of 2-5cm and the length of 20-100cm, and the chitosan with different deacetylation degrees is intercepted by different ion exchange columns, so that the aim of separating the chitosan with different deacetylation degrees is fulfilled.

3. Liquid nitrogen quenching in the process of preparing the nerve conduit is beneficial to the uniform dispersion of insoluble chitin in injection molding liquid, but vitrification freezing can be formed, and crystallization and volume expansion are avoided by vitrification freezing. The molecules that make up the material are solidified in their original positions and water molecules are difficult to escape during sublimation. Is very disadvantageous for lyophilization. Therefore, before the freeze-drying step, the injected mould is placed below the eutectic point of the chitosan solution and heated for recrystallization so as to eliminate vitrification crystallization. During the forming process of the catheter, the alkaline solution is used for neutralizing the residual weak acid in the catheter so as to improve the strength of the catheter.

4. According to the invention, the acidic amino acid and dopamine are added into the chitosan, so that the self-adhesion of the nerve conduit is realized, the manual suture operation is omitted, and the treatment efficiency of medical workers is improved. Wherein, carboxyl in the acidic amino acid can simultaneously form amido bond with chitosan and amino in human nerves, thereby realizing the self-connection of the nerve conduit and the nerves; meanwhile, dopamine can form polydopamine with high biological adhesion in an alkaline solution, and the polydopamine and an amido bond together improve the adhesion between the nerve conduit and the nerve. In addition, the dopamine is oxidized and polymerized by self under the action of alkaline solution to form polydopamine which has good biocompatibility and degradation characteristic and can not influence the normal use of the nerve conduit.

5. The nerve conduit provided by the invention can be used independently, and can also be used together with a PGLA (poly-beta-lactide) fibrous scaffold. When the PGLA fiber scaffold is used, guiding and climbing conditions are provided for nerves, the mechanical strength and toughness of the catheter and the cell growth space inside the catheter are not influenced, the catheter can be used independently when the damaged nerves need to be repaired simply, and the PGLA fiber scaffold can be matched with the scaffold when the nerve bundles with complex nerve structures are more. The catheter does not contain exogenous toxic and side effect substances substituted by a preparation process, has high porosity and provides a necessary way for transporting nutrient substances required by the growth of nerve cells. Meanwhile, the mechanical strength of the catheter is high, and the catheter can adapt to various stress conditions after transplantation.

Drawings

FIG. 1 is a graph showing the degradation of nerve conduits prepared in examples 1 to 3 with time;

FIG. 2 is a scanning electron micrograph of a nerve conduit prepared in example 1;

FIG. 3 is a scanning electron micrograph of a nerve conduit prepared in example 3.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.

Example 1

This example provides a short-term nerve conduit with a degradation time of 20 days, with a porosity of 80% and a degree of deacetylation of the starting chitosan of 70%.

The preparation method of the nerve conduit comprises the following steps:

(1) Dissolving 250g of refined chitosan by using 100L of 3% acetic acid, sequentially passing through 8 CM sepharose CL-6B ion exchange columns with the diameter of 4.5CM and the length of 80CM, taking out a 7 th chromatographic column, eluting by using acetic acid-sodium acetate buffer solution with the pH of 5.6 and containing 0.5mol/L NaCl, adding 15L of NaOH solution with the mass concentration of 30% into eluent, washing precipitates by using distilled water, and drying to obtain chitosan with the deacetylation degree of 70%;

(2) 10g of L-glutamic acid is dissolved in 650 mM MES buffer solution (2.5 mM, pH6.0, MES buffer solution pH is adjusted to 8.0, and after L-glutamic acid is dissolved, the MES buffer solution pH is adjusted to 6.0), 3.5g of N-hydroxysuccinimide and 5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are added, and after 2 hours of activation, activated L-glutamic acid is obtained;

(3) Dissolving 25g of chitosan with deacetylation degree of 70% by using 120mL of 1% acetic acid, adding 2g of L-glutamic acid activated in the step (2) and 6g of dopamine, reacting at room temperature for 24h, injecting into a mold, quickly freezing by using liquid nitrogen, placing the mold into a refrigerator with the temperature of-20 ℃, after 5h of devitrification, freeze-drying for 16h at the temperature of-30 ℃ and the vacuum degree of 180Pa, solidifying and molding the liquid, neutralizing the residual weak acid by using 25mL of NaOH solution with the mass concentration of 30%, cleaning by using distilled water, freeze-drying again at the temperature of-30 ℃ and the vacuum degree of 180Pa, and obtaining the nerve conduit with degradation time of 20 days, wherein a scanning electron microscope picture of the nerve conduit is shown in figure 2.

Example 2

This example provides a mid-stage nerve conduit with a degradation time of 39 days, with a porosity of 72% and a degree of deacetylation of the starting chitosan of 88%.

The preparation method of the nerve conduit comprises the following steps:

(1) Dissolving 100g of refined chitosan by using 40L of 1% acetic acid, sequentially passing through 7 CM sepharose CL-6B ion exchange columns with the diameter of 4.5CM and the length of 80CM, taking out the 4 th chromatographic column, eluting by using acetic acid-sodium acetate buffer solution with the pH value of 5.6 and containing 0.1mol/L NaCl, adding 6L of KOH solution with the mass concentration of 30% into eluent for treatment, washing precipitates by using distilled water, and drying to obtain the chitosan with the deacetylation degree of 88%;

(2) Dissolving 7g of aspartic acid in 0.4LMES buffer solution (2.5 mM, pH6.0, adjusting the pH of MES buffer solution to 8.0, adjusting the pH to 6.0 after the aspartic acid is dissolved), adding 3.3g of N-hydroxysuccinimide and 4.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating for 2.5h to obtain activated aspartic acid;

(3) Dissolving 10g of chitosan with deacetylation degree of 88% by using 33mL of 1% acetic acid, adding 0.7g of activated aspartic acid and 0.2g of dopamine, reacting at room temperature for 24h, injecting into a mold, quickly freezing by using liquid nitrogen, putting the mold into a refrigerator with the temperature of-10 ℃, performing devitrification for 10h, performing freeze-drying for 24h at the temperature of-20 ℃ and the vacuum degree of 200Pa, and curing and forming liquid; then, 100mL of KOH solution with the mass concentration of 30% is used for neutralizing the residual weak acid, and then the residual weak acid is washed by distilled water, and then the neural catheter is freeze-dried for 24 hours under the conditions of the temperature of minus 20 ℃ and the vacuum degree of 200Pa, so that the neural catheter with the degradation time of 39 days is obtained.

Example 3

This example provides a long-term nerve conduit with a degradation time of 151 days, with a porosity of 70% and a degree of deacetylation of the starting chitosan of 98%.

The preparation method of the nerve conduit comprises the following steps:

(1) Dissolving 500g of refined chitosan by 200L of 1% acetic acid, sequentially passing through 10 CM sepharose CL-6B ion exchange columns with the diameter of 4.5CM and the length of 80CM, taking out the 1 st chromatographic column, eluting by an acetic acid-sodium acetate buffer solution with the pH of 5.6 and containing 2mol/L NaCl, adding 30L of NaOH solution with the mass concentration of 30% into eluent, washing the precipitate by distilled water, and drying to obtain the chitosan with the deacetylation degree of 98%;

(2) Dissolving 9g of L-glutamic acid in 0.5LMES buffer solution (2.5 mM, pH6.0, adjusting the pH of MES buffer solution to 8.0, adjusting the pH to 6.0 after the L-glutamic acid is dissolved), adding 2.3g of N-hydroxysuccinimide and 3.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and activating for 3 hours to obtain activated L-glutamic acid;

(3) Dissolving 50g of chitosan with deacetylation degree of 98% by using 165mL of 1% acetic acid, adding 4.5g of activated L-glutamic acid obtained in the step (2) and 15g of dopamine, reacting at room temperature for 24h, injecting into a mold, quickly freezing by using liquid nitrogen, placing the mold into a refrigerator with the temperature of-30 ℃, performing devitrification for 1h, performing freeze-drying at the temperature of-100 ℃ for 12h, solidifying and molding the liquid, neutralizing the residual weak acid by using 50mL of NaOH solution with the mass concentration of 30% to improve the strength of the catheter, washing with distilled water, and performing freeze-drying at the temperature of-100 ℃ for 12h to obtain the nerve catheter with degradation time of 151 days, wherein a scanning electron microscope picture is shown in figure 3.

Example 4

This example provides a short-term nerve conduit with a degradation time of 21 days, with a porosity of 77% and a degree of deacetylation of the starting chitosan of 70%.

Compared with example 1, the preparation method is different only in that L-glutamic acid and dopamine are omitted, and deacetylated chitosan is directly used for preparing the nerve conduit.

Example 5

This example provides a short-term nerve conduit with a degradation time of 23 days, with a porosity of 82% and a degree of deacetylation of the starting chitosan of 70%.

The preparation method is different from that of example 1 only in that L-glutamic acid is omitted, deacetylated chitosan is dissolved in 1% acetic acid, 30g of dopamine is added to react for 24 hours, and injection molding is performed to prepare the nerve conduit.

Example 6

This example provides a short-term nerve conduit with a degradation time of 19 days, with a porosity of 81% and a degree of deacetylation of the starting chitosan of 70%.

The preparation method differs from example 1 only in omitting dopamine.

Comparative example 1

This comparative example provides a nerve conduit with a degradation time of 6 days, with a porosity of 85% and a degree of deacetylation of the starting material chitosan of 65%.

The preparation method of the nerve conduit comprises the following steps:

(1) Dissolving 250g of refined chitosan by using 100L of 1% acetic acid, sequentially passing through 9 CM sepharose CL-6B ion exchange columns with the diameter of 4.5CM and the length of 80CM, taking out the 8 th chromatographic column, eluting by using acetic acid-sodium acetate buffer solution with the pH of 5.6 and containing 0.5mol/L NaCl, adding 15L of NaOH solution with the mass concentration of 30% into eluent, washing precipitates by using distilled water, and drying to obtain the chitosan with the deacetylation degree of 65%;

(2) 10g of L-glutamic acid was dissolved in 650 mM MES buffer solution (2.5 mM, pH 6.0), and 3.5g of N-hydroxysuccinimide and 5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added thereto to activate for 2 hours, thereby obtaining activated L-glutamic acid;

(3) Dissolving 25g of chitosan with deacetylation degree of 70% by using 120mL of 1% acetic acid, adding 2g of activated L-glutamic acid obtained in the step (2) and 6g of dopamine, reacting at room temperature for 24 hours, injecting into a mold, quickly freezing by using liquid nitrogen, placing the mold into a refrigerator with the temperature of-20 ℃, after 5 hours of devitrification, freeze-drying for 16 hours at the temperature of-30 ℃ and under the vacuum degree of 180Pa, solidifying and forming liquid, neutralizing residual weak acid by using 25mL of NaOH solution with mass concentration of 30%, washing with distilled water, and freeze-drying again at the temperature of-30 ℃ and under the vacuum degree of 180Pa to obtain the nerve conduit with degradation time of 6 days.

Comparative example 2

This comparative example provides a nerve conduit with unstable degradation time, the porosity of which was 78%, the raw material chitosan was not deacetylated.

The preparation method of the nerve conduit comprises the following steps:

(1) 10g of L-glutamic acid was dissolved in 650LMES buffer solution (2.5 mM, pH 6.0), and 3.5g of N-hydroxysuccinimide and 5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were added thereto to activate for 2 hours, thereby obtaining activated L-glutamic acid;

(2) Dissolving 25g of chitosan by using 120mL of 1% acetic acid, adding 2g of L-glutamic acid activated in the step (1) and 6g of dopamine, reacting at room temperature for 24h, injecting into a mold, quickly freezing by using liquid nitrogen, placing the mold into a refrigerator at the temperature of-20 ℃, performing glass removal for 5h, performing freeze-drying for 16h at the temperature of-30 ℃ and under the vacuum degree of 180Pa, solidifying and molding the liquid, neutralizing the residual weak acid by using 25mL of NaOH solution with the mass concentration of 30%, washing with distilled water, and performing freeze-drying again at the temperature of-30 ℃ and under the vacuum degree of 180Pa to obtain the nerve conduit.

Experimental example 1

Examples 1-6 nerve conduits were tested for porosity by the following specific method:

1. adding absolute ethyl alcohol into the test tube, and recording the volume as V ₀ ；

2. The nerve conduits of examples 1 to 6 and comparative examples 1 to 2 were placed in test tubes, respectively, and when the catheters were completely soaked in absolute ethanol and no air bubbles were present, the total volume of the catheters and absolute ethanol was designated as V ₁ Volume difference V ₁ -V ₀ Represents the volume of the scaffold material profile, i.e., the volume of material excluding pores;

3. the nerve conduit was removed from the test tube and the volume of absolute ethanol remaining was recorded as V ₂ ；

4. Calculating the porosity: p (%) = [ (V) ₀ -V ₂ )/(V ₁ -V ₂ )]×100％

Wherein: v ₀ -V ₂ Represents the volume of absolute ethanol impregnated into the scaffold material, i.e., the volume of the pores of the scaffold material; v ₁ -V ₂ The total volume of the scaffold material is represented and the specific results are shown in table 1.

TABLE 1 porosity of nerve conduits

	Porosity (%)
		Example 1	80
Example 2	72
		Example 3	70
Example 4	77
		Example 5	82
Example 6	81

The data in table 1 show that the nerve conduit provided by the invention has high porosity and provides a necessary way for transporting nutrients required by nerve cell growth.

Experimental example 2

Examining the degradation time of the nerve conduits of examples 1-6 and comparative examples 1-2, the specific method is as follows: 3 nerve conduits of examples 1 to 6 and comparative examples 1 to 2, each having a length of about 10mm, were each taken out and put into a centrifuge tube, 5ml of a physiological saline solution containing lysozyme (4 mg/ml) was added to each tube, and the tube was placed in a 37 ℃ constant temperature water bath cabinet, and the freshly prepared enzyme solution was replaced every 24 hours, and the time required for complete degradation of each nerve conduit was observed, and the results are shown in Table 2 and FIG. 1.

TABLE 2 degradation time of nerve conduits

The data in table 2 show that when the degree of deacetylation is very small (comparative example 1), or when chitosan is not deacetylated (comparative example 2), uncertainty occurs in the degradation time of the nerve conduit, the degradation time of the nerve conduit cannot be determined by the degree of deacetylation of the chitosan as a raw material, and prediction of the degradation time of the nerve conduit and medical application are inconvenient.

FIG. 1 shows that the degradation time of the nerve conduit can be controlled by controlling the deacetylation degree of chitosan as a raw material, and the conduits with different degradation times can be selected according to nerve injury conditions, so that the nerve conduit is prevented from being degraded too fast, nerves are not grown completely and support is not lost; meanwhile, the condition that the nerve is recovered and the catheter is not degraded for a long time to cause inflammation can be avoided. Meanwhile, the data in table 2 also show that the degradation time of the nerve conduit is not affected by the preparation of the nerve conduit by chitosan and acidic amino acid and/or dopamine together and the preparation of the nerve conduit by chitosan alone.

Experimental example 3

Rat sciatic nerves were brought into contact with both ends of the nerve conduits of examples 1 to 6 of the present invention, fixed under a humid condition at 37 ℃ for 10min, and then the adhesion of the nerve conduits to the nerves was tested using a universal mechanical testing machine at a crosshead speed of 0.5mm/min, with the test results shown in Table 3.

TABLE 3 autohension of nerve conduits to nerves

The data in table 3 show that the nerve conduit provided by the invention has autohension with nerves, so that the step of manual sewing can be omitted, wherein in example 4, the use of acidic amino acid and dopamine is omitted, and the autohension of the nerve conduit with nerves is obviously reduced; examples 5 and 6, which omit the acidic amino acid and dopamine, respectively, also have low autohesion of nerve conduits to nerves.

The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (2)

1. A chitosan nerve conduit is characterized in that the nerve conduit is divided into a short-term conduit, a medium-term conduit and a long-term conduit; wherein the short-term catheter degradation time is 7-30 days, the porosity is 75-85%, and the deacetylation degree of the raw material chitosan: DD is between 70% and 75%; the medium-term catheter degradation time is 31-90 days, the porosity is 70-75%, and the deacetylation degree of the raw material chitosan: DD is more than 75 percent and less than or equal to 90 percent; the degradation time of the long-term catheter is 91-200 days, the porosity is 65-70%, and the deacetylation degree of the raw material chitosan is more than 90% and less than or equal to DD and 98%;

the preparation method of the chitosan nerve conduit comprises the following steps:

(1) Dissolving chitosan, separating by 1-10 ion exchange columns with diameter of 2-5cm and length of 20-100cm, eluting, treating with alkaline solution, and drying to obtain chitosan;

(2) Dissolving chitosan, then performing injection molding, freezing, devitrifying, curing and forming, treating with alkaline solution, and freeze-drying to obtain the nerve conduit;

the step (2) of injection molding further comprises a step of mixing chitosan with an acidic amino acid and dopamine; the acidic amino acid is selected from one or two of glutamic acid and aspartic acid;

the mass ratio of the chitosan to the acidic amino acid to the dopamine is (100);

the step of activating the acidic amino acid by a catalyst is also included before the step of mixing the chitosan with the acidic amino acid and the dopamine; the catalyst is selected from one or two of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the mass ratio of the acidic amino acid to the catalyst is 10-12.

2. The nerve conduit according to claim 1, wherein any one of the following is also satisfied:

(A) In the step (2), the glass removing temperature is between-10 ℃ and-30 ℃ and the time is 1-10h;

(B) In the step (2), the forming method is freeze drying, the temperature is-20 ℃ to-100 ℃, the vacuum degree is 0-200Pa, and the time is 12-24h;

(C) The ion exchange column in the step (1) is selected from one or more of a sephadex column and an agarose gel column;

(D) The eluent in the step (1) is acetic acid-sodium acetate buffer solution containing 0.1-2mol/L NaCl;

(E) The deacetylation degree of the chitosan obtained in the step (1) is 70-98%;

(F) In the step (1) and/or the step (2), the alkaline solution is selected from one or two of NaOH and KOH solution;

(G) The mass percentage concentration of the alkaline solution is 10-80%.

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