CN110448727B - Adhesive hydrogel material, suture-free artificial nerve conduit and preparation method thereof - Google Patents
Adhesive hydrogel material, suture-free artificial nerve conduit and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of medical biomaterials capable of being implanted into a human body, and discloses a viscous hydrogel material, a suture-free artificial nerve conduit and a preparation method thereof. The invention combines the viscous hydrogel material with the tissue adhesion function and the chitosan artificial nerve conduit, and provides the suture-free artificial nerve conduit which is free from manual operation suture and convenient to use. The viscous hydrogel material bonded on the end side of the suturing-free artificial nerve conduit has good histocompatibility and mechanical property, can be strongly bonded with the wet tissue surface through physical action and chemical bonding, and can avoid the complicated suturing process when the conduit is implanted in an operation. In order to prepare the suture-free artificial nerve conduit, the invention also discloses a preparation method which is simple and feasible and is suitable for production.
Description
Technical Field
The invention belongs to the field of medical biomaterials capable of being implanted into a human body, and particularly relates to a viscous hydrogel material, a suture-free artificial nerve conduit and a preparation method thereof.
Background
In China, the number of peripheral nerve injury patients tends to increase year by year. Although peripheral nerves have a certain self-repair function, complete self-repair, particularly long-distance nerve defects, is difficult to achieve, and thus repair by means of various nerve bridges is required. Although autologous scaffolds are the gold standard for repairing peripheral nerve defects, they suffer from limited donor source, size mismatch and permanent damage to the site of extraction. At present, people mainly realize nerve repair by means of various tissue engineering nerve bridges prepared from natural or synthetic biomaterials with better biocompatibility and biodegradability. However, the speed of these artificial nerve grafts to promote peripheral nerve regeneration is still difficult to meet clinical needs, especially to repair nerve defects over long distances. Therefore, the development of a novel nerve graft which can promote peripheral nerve regeneration quickly and realize long-distance nerve defect repair is of great scientific significance.
Hydrogels are highly cross-linked hydrophilic polymers, structurally similar to the natural extracellular matrix, with good biocompatibility, have been widely studied, and have been applied in the medical field. Naturally occurring polymers such as chitosan, alginate, Hyaluronic Acid (HA), collagen and gelatin, etc., have biodegradability but suffer from poor mechanical properties, immunogenicity and batch-to-batch variability. While synthetic polymers such as polyethylene glycol (PEG), Polyacrylamide (PAM), poly (vinyl alcohol) (PVA), and Polymethylmethacrylate (PMMA) generally have better mechanical properties and low immunogenicity, but lack biological functions. Therefore, the hydrogel formed by compounding natural and synthetic polymers can meet the application in vivo.
Adhesion to wet and dynamic surfaces, including biological tissue, is important in many areas and adhesives that can strongly adhere to biological tissue will have a wide range of applications, from tissue repair and drug delivery. However, existing tissue adhesives are far from ideal, or are cytotoxic, poorly tissue compatible, or have poor adhesion to moist tissue surfaces. Therefore, there is an urgent need to develop biomaterials having good adhesion and tissue compatibility on wet tissue surfaces.
Disclosure of Invention
In view of the above, the present invention aims to provide a viscous hydrogel material, a suture-free artificial nerve conduit and a preparation method thereof. The adhesive hydrogel material can realize the adhesion of the artificial nerve conduit and the surface of the wet and dynamic tissue, does not need to be sutured, and is convenient to use.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method of preparing a viscous hydrogel material, comprising:
1) dissolving chitosan, sodium alginate and acrylamide in MES buffer solution to obtain mixed solution;
2) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the mixed solution, stirring and mixing uniformly, and then adding N, N' -methylene bisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO4And stirring for 0.5-1 h at room temperature to obtain the viscous hydrogel material.
Preferably, the MES buffer has a pH of 5.
Preferably, the content of each component in the mixed solution is as follows:
more preferably, the content of each component in the mixed solution is as follows:
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.
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 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.
7) And (4) disassembling the die, and soaking and washing in distilled water to remove the uncrosslinked cross-linking agent and acrylamide.
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.
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 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.
7) And (4) disassembling the die, and soaking and washing in distilled water to remove the uncrosslinked cross-linking agent and acrylamide.
Comparative 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) 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.
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 18h 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.
Comparative example 2
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) 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.
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 40h 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.
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 (4)
1. A preparation method of a suture-free artificial nerve conduit is characterized by comprising the following steps:
(1) dissolving chitosan, sodium alginate and acrylamide in MES buffer solution to obtain mixed solution; the mixed solution comprises the following components in percentage by weight: 20g/L of chitosan; sodium alginate 20 g/L; 120g/L of acrylamide; the pH value of the MES buffer solution is 5;
(2) adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the mixed solution, stirring and mixing uniformly, and then adding N, N' -methylene bisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO4Stirring for 0.5-1 h at room temperature to obtain a viscous hydrogel material;
(3) 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;
(4) 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.
2. The method for preparing a suture-free artificial nerve conduit as claimed in claim 1, wherein the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (2) is used 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。
3. The method for preparing a suture-free artificial nerve conduit according to claim 1, wherein the mold is matched with the inner diameter and the outer diameter of the chitosan artificial nerve conduit.
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.
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| CN111450318B (en) * | 2020-03-26 | 2022-05-17 | 南通大学 | Chitosan nerve conduit with illumination bridged nerve tissue and preparation method and application thereof |
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