preferably, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride used in the step (2) is added in an amount of 0.12g per 1mL of the mixed solution; the using amount of the N-hydroxysuccinimide is 0.12g per 1mL of the mixed solution; the using amount of the N, N '-methylene-bisacrylamide is that 3.6 mu L of N, N' -methylene-bisacrylamide with the concentration of 20g/L is added into each 1mL of the mixed solution; the usage amount of the tetramethylethylenediamine is that 0.8 mu L of the tetramethylethylenediamine is added into each 1mL of the mixed solution; the dosage of the ammonium persulfate is 22.6 mu L of ammonium persulfate with the concentration of 0.27M added into each 1mL of the mixed solution; CaSO₄In an amount of 19. mu.L of 0.75M CaSO per 1mL of the mixed solution₄。

According to the invention, N' -methylene bisacrylamide is added as a cross-linking agent during preparation of the viscous hydrogel, and in a redox initiation system consisting of tetramethylethylenediamine and ammonium persulfate, free radical polymerization reaction is carried out on acrylamide to form polyacrylamide, and activated hydroxyl in sodium alginate can form an amido bond with polyacrylamide, so that the mechanical property of the chitosan hydrogel can be enhanced, and the chitosan hydrogel can be rapidly molded in the preparation process. In addition, CaSO₄The calcium ion in the solution is coupled with the alginic acid in the sodium alginate solution to form a cross-linked network, and the viscous crystal glue material can be quickly molded in a mold.

The invention also provides a viscous hydrogel material prepared by the preparation method.

The adhesive hydrogel is of a porous structure, has no cytotoxicity, is favorable for exchange of nutrient substances inside and outside the nerve conduit, cell migration and tissue regeneration, and has strong histocompatibility.

The invention also provides a suture-free artificial nerve conduit, which comprises a chitosan artificial nerve conduit and adhesive hydrogel respectively adhered to the two end parts of the chitosan artificial nerve conduit, wherein the adhesive hydrogel is formed by gelatinizing the adhesive hydrogel material.

The invention also provides a preparation method of the suture-free artificial nerve conduit, which comprises the following steps:

(1) placing the chitosan artificial nerve conduit into a mould, and reserving vacant spaces with the lengths of 1-2 cm at two ends of the mould;

(2) injecting the viscous hydrogel material to two ends of the chitosan artificial nerve conduit placed in the mold before gelling, placing for 12-24 h, and removing the mold after the viscous hydrogel material naturally gels to obtain the suture-free artificial nerve conduit.

Preferably, the mold is matched with the inner diameter and the outer diameter of the chitosan artificial nerve conduit.

Preferably, the length of the chitosan artificial nerve conduit is 5 mm.

The present invention also provides a suture-free artificial nerve conduit characterized by being prepared by adhering the adhesive porous hydrogel of claim 1 or 2 to both ends of a chitosan artificial nerve conduit, respectively.

Compared with the prior art, in the preparation method of the viscous hydrogel disclosed by the invention, chitosan, sodium alginate and acrylamide are mixed, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added and mixed uniformly, and then N, N' -methylenebisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO are added₄The obtained viscous hydrogel material has a porous structure, is free from cytotoxicity, has good biocompatibility and is suitable for being implanted into a body. In addition, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide added in the preparation process of the viscous hydrogel material can activate carboxyl in sodium alginate, and then can form amido bond with chitosan and amino in human tissues to form a cross-linked network. Therefore, the viscous hydrogel material is injected to the positions of two ends in a mould with the chitosan artificial nerve conduit before gelling, hydroxyl in sodium alginate forms amido bond with chitosan, and the chitosan artificial nerve conduit is strongly bonded and molded at the position of the end of the chitosan nerve conduit to prepare the suture-free artificial nerve conduit. When the suturing-free artificial nerve conduit is implanted in a use process, such as a nerve conduit operation, the viscous hydrogel can strongly adhere to nerve tissues by forming an amido bond with amino in the nerve tissues, so that the effect of avoiding operation suturing is achieved, and the use is convenient.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow.

All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.

Example 1

1) 1L of MES buffer was prepared and the pH was adjusted to 5.

2) And (3) weighing 20g of chitosan, 20g of sodium alginate and 120g of acrylamide, and dissolving in the prepared MES buffer solution to obtain a mixed solution.

3) 120g of amino crosslinker 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 120g of N-hydroxysuccinimide (NHS) are added to the mixed solution, after mixing, 3.6mL of a covalent crosslinking agent N, N' -Methylenebisacrylamide (MBAA) with a concentration of 20g/L, 0.8mL of an accelerator Tetramethylethylenediamine (TEMED), 22.6mL of an initiator Ammonium Persulfate (APS) with a concentration of 0.27M and 19.1mL of an ionic crosslinking agent CaSO with a concentration of 0.75M are added₄And uniformly mixing to obtain the viscous hydrogel material.

5) The chitosan artificial nerve conduit is placed into a plastic mould with matched inner and outer diameters, and 2cm of space is reserved at two ends.

6) Before the viscous hydrogel material is not gelatinized, the viscous hydrogel material is injected into the vacant spaces at the two ends of the chitosan catheter, and is placed for 12 hours to be gelatinized naturally.

7) And (4) disassembling the die, and soaking and washing in distilled water to remove the uncrosslinked cross-linking agent and acrylamide.

Example 2

1) 1L of MES buffer was prepared and the pH was adjusted to 5.

2) 30g of chitosan, 30g of sodium alginate and 120g of acrylamide are weighed and dissolved in the prepared MES buffer solution to obtain a mixed solution.

6) Before the hydrogel is not gelatinized, the hydrogel is injected into the vacant spaces at the two ends of the chitosan catheter, and the chitosan catheter is placed for 18h to naturally gelatinize.

Example 3

1) 1L of MES buffer was prepared and the pH was adjusted to 5.

2) 10g of chitosan, 10g of sodium alginate and 70g of acrylamide are weighed and dissolved in the prepared MES buffer solution to obtain a mixed solution.

3) 120g of amino crosslinker 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 120g of N-hydroxysuccinimide (NHS) are added to the mixed solution, after mixing, 3.6mL of a covalent crosslinking agent N, N' -Methylenebisacrylamide (MBAA) with a concentration of 20g/L, 0.8mL of an accelerator Tetramethylethylenediamine (TEMED), 22.6mL of an initiator Ammonium Persulfate (APS) with a concentration of 0.27M and 19.1mL of an ionic crosslinking agent CaSO with a concentration of 0.75M are added₄Mixing to obtain viscous hydrogelA material.

6) Before the hydrogel is not gelatinized, the hydrogel is injected into the vacant spaces at the two ends of the chitosan catheter, and the chitosan catheter is placed for 24 hours to naturally gelatinize.

Comparative example 1

1) 1L of MES buffer was prepared and the pH was adjusted to 5.

3) 120g of amino crosslinker 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 120g of N-hydroxysuccinimide (NHS) were added to the mixed solution, and after mixing, 0.8mL of accelerator Tetramethylethylenediamine (TEMED), 22.6mL of initiator Ammonium Persulfate (APS) having a concentration of 0.27M and 19.1mL of ionic crosslinker CaSO having a concentration of 0.75M were added₄And uniformly mixing to obtain the viscous hydrogel material.

6) Before the viscous hydrogel material is not gelatinized, the viscous hydrogel material is injected into the vacant spaces at the two ends of the chitosan catheter, and is placed for 18h to be gelatinized naturally.

Comparative example 2

1) 1L of MES buffer was prepared and the pH was adjusted to 5.

3) 120g of amino crosslinker 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 120g of N-hydroxysuccinimide (NHS) were added to the mixed solution, and after mixing, 3.6mL of covalent crosslinker N, N' -Methylenebisacrylamide (MBAA) with a concentration of 20g/L was added) 0.8mL of accelerator Tetramethylethylenediamine (TEMED) and 19.1mL of 0.75M ionic crosslinker CaSO₄And uniformly mixing to obtain the viscous hydrogel material.

6) Before the viscous hydrogel material is not gelatinized, the viscous hydrogel material is injected into the vacant spaces at the two ends of the chitosan catheter, and is placed for 40h to be gelatinized naturally.

Test example: adhesive hydrogel adhesion test

Rat sciatic nerves were inserted into 1mm of the adhesive hydrogel at both ends of the suture-free artificial nerve conduit prepared in examples 1-3 of the present invention, and fixed with surgical forceps under a humid condition at 37 ℃ for 10 min. The test was performed using a universal mechanical testing machine (TFW-58, Shanghai Tuofeng Instrument Technology co.ltd., China) at a crosshead speed of 0.5mm/min, with the test results shown in table 1.

TABLE 1 adhesion of artificial nerve conduits to nerves

According to the test results in Table 1, the adhesive force of the adhesive hydrogels at two ends of the suturing-free artificial nerve conduit provided by the embodiment and the comparative example is 0.01-0.1 MPa, wherein the adhesive force of the adhesive hydrogels at two ends of the suturing-free artificial nerve conduit prepared by the embodiment 1 (the concentration is 20g/L of chitosan; 20g/L of sodium alginate; 20g/L of acrylamide; 120g/L) is the strongest to nerve, and can reach 0.1 MPa. In addition, it was found according to the comparative example that removal of Tetramethylethylenediamine (TEMED) resulted in a decrease in adhesive force of the adhesive porous hydrogel, and that removal of Ammonium Persulfate (APS) resulted in substantially no decrease in adhesive force of the adhesive hydrogel, but resulted in an increase in gel formation time.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

Translated fromChinese

1.一种免缝合人工神经导管的制备方法，其特征在于，包括以下步骤：1. a preparation method of suture-free artificial nerve conduit, is characterized in that, comprises the following steps:

(1)将壳聚糖、海藻酸钠和丙烯酰胺溶于MES缓冲液，得混合溶液；所述混合溶液中各组分含量为：壳聚糖20g/L；海藻酸钠20g/L；丙烯酰胺120g/L；所述MES缓冲液的pH值为5；(1) Dissolve chitosan, sodium alginate and acrylamide in MES buffer to obtain a mixed solution; the content of each component in the mixed solution is: chitosan 20g/L; sodium alginate 20g/L; propylene Amide 120g/L; the pH value of the MES buffer is 5;

(2)向所述混合溶液中加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺，搅拌混合均匀后，再加入N，N'-亚甲基双丙烯酰胺、四甲基乙二胺、过硫酸铵和CaSO₄，室温下搅拌0.5～1h，得粘性水凝胶材料；(2) Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the mixed solution, stir and mix well, then add N , N'-methylenebisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO₄ , stirring at room temperature for 0.5-1 h to obtain a viscous hydrogel material;

(3)将壳聚糖人工神经导管放入模具中，所述模具两端留出1～2cm长度的空余空间；(3) putting the chitosan artificial nerve conduit into the mold, leaving a free space of 1-2 cm in length at both ends of the mold;

(4)所述粘性水凝胶材料在成胶之前注射到放置在所述模具中的所述壳聚糖人工神经导管的两端，放置12～24h，待所述粘性水凝胶材料自然成胶后，卸掉模具，得免缝合人工神经导管。(4) The viscous hydrogel material is injected into the two ends of the chitosan artificial nerve conduit placed in the mold before gelation, and placed for 12-24 hours, and the viscous hydrogel material is naturally formed. After the glue is applied, the mold is removed to avoid suturing the artificial nerve conduit.

2.根据权利要求1所述的免缝合人工神经导管的制备方法，其特征在于，步骤(2)中1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐的使用量为每1mL的所述混合溶液中加入0.12g；N-羟基琥珀酰亚胺的使用量为每1mL的所述混合溶液中加入0.12g；N，N'-亚甲基双丙烯酰胺的使用量为每1mL的所述混合溶液中加入3.6μL浓度为20g/L的N，N'-亚甲基双丙烯酰胺；四甲基乙二胺的使用量为每1mL的所述混合溶液中加入0.8μL的四甲基乙二胺；过硫酸铵的用量为每1mL的所述混合溶液中加入22.6μL的浓度0.27M的过硫酸铵；CaSO₄的用量为每1mL的所述混合溶液中加入19μL的浓度为0.75M的CaSO₄。2. the preparation method of suture-free artificial nerve conduit according to claim 1, is characterized in that, in step (2), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride The usage amount is 0.12g per 1mL of the mixed solution; the usage amount of N-hydroxysuccinimide is 0.12g per 1mL of the mixed solution; N,N'-methylenebisacrylamide The usage amount is to add 3.6 μL of N,N'-methylenebisacrylamide with a concentration of 20g/L in every 1mL of the mixed solution; the usage amount of tetramethylethylenediamine is per 1mL of the mixed solution Add 0.8 μL of tetramethylethylenediamine; the dosage of ammonium persulfate is 22.6 μL of 0.27M ammonium persulfate per 1 mL of the mixed solution; the dosage of CaSO₄ is per 1 mL of the mixed solution 19 μL of 0.75 M CaSO₄ was added to the solution.

3.根据权利要求1所述的免缝合人工神经导管的制备方法，其特征在于，所述模具与所述壳聚糖人工神经导管内外径匹配。3 . The method for preparing a suture-free artificial nerve conduit according to claim 1 , wherein the mold matches the inner and outer diameters of the chitosan artificial nerve conduit. 4 .

4.根据权利要求1所述的免缝合人工神经导管的制备方法，其特征在于，所述壳聚糖人工神经导管的长度为5mm。4 . The method for preparing a suture-free artificial nerve conduit according to claim 1 , wherein the length of the chitosan artificial nerve conduit is 5 mm. 5 .