技术领域technical field
本发明属于可植入人体中的医用生物材料领域，尤其涉及一种粘性水凝胶材料、免缝合人工神经导管及其制备方法。The invention belongs to the field of medical biomaterials that can be implanted into human bodies, and in particular relates to a viscous hydrogel material, a suture-free artificial nerve guide and a preparation method thereof.
背景技术Background technique
在我国，周围神经损伤患者数量呈逐年增多的趋势。尽管周围神经具有一定的自我修复功能，但难以实现完全自我修复特别是长距离神经缺损，因此需要借助于各种神经桥接物进行修复。虽然自体同源支架是修复周围神经缺损的金标准，但是却面临着供体来源有限，尺寸不匹配以及会给取材部位造成永久伤害等问题。目前，人们主要借助由具有较好生物相容性和生物降解性的天然或合成生物材料制备而成的各种组织工程神经桥接物来实现神经修复。但这些人工神经移植物促进周围神经再生的速度仍然难以满足临床需要，特别是难以修复较长距离的神经缺损。因此，开发能较快促进周围神经再生并能实现较长距离神经缺损修复的新型神经移植物具有非常重要的科学意义。In my country, the number of patients with peripheral nerve injury is increasing year by year. Although peripheral nerves have a certain self-repair function, it is difficult to achieve complete self-repair, especially for long-distance nerve defects, so it needs to be repaired with the help of various nerve bridges. Although autologous scaffolds are the gold standard for repairing peripheral nerve defects, they face problems such as limited donor sources, mismatched sizes, and permanent damage to the harvested site. At present, people mainly rely on various tissue-engineered nerve bridges prepared from natural or synthetic biomaterials with good biocompatibility and biodegradability to achieve nerve repair. However, the speed at which these artificial nerve grafts promote peripheral nerve regeneration is still difficult to meet clinical needs, especially for repairing long-distance nerve defects. Therefore, it is of great scientific significance to develop new nerve grafts that can rapidly promote peripheral nerve regeneration and achieve long-distance nerve defect repair.
水凝胶是高度交联的亲水聚合物，结构与天然细胞外基质的相似，具有良好生物相容性，已被广泛研究，并且已经在医学领域中应用。天然存在的聚合物如壳聚糖，藻酸盐，透明质酸(HA)，胶原和明胶等，具有可生物降解性，但存在机械性能差、免疫原性以及批次间的差异性等缺点。而聚乙二醇(PEG)、聚丙烯酰胺(PAM)、聚(乙烯醇)(PVA)和聚甲基丙烯酸甲酯(PMMA)等合成类聚合物一般具有更良好的机械性能和低免疫原性，但缺乏生物功能。因此由天然和合成类聚合物复合形成的水凝胶可以满足体内应用。Hydrogel is a highly cross-linked hydrophilic polymer with a structure similar to that of natural extracellular matrix and has good biocompatibility. It has been widely studied and has been applied in the medical field. Naturally occurring polymers such as chitosan, alginate, hyaluronic acid (HA), collagen, and gelatin are biodegradable but suffer from disadvantages such as poor mechanical properties, immunogenicity, and batch-to-batch variability . Synthetic polymers such as polyethylene glycol (PEG), polyacrylamide (PAM), poly(vinyl alcohol) (PVA) and polymethyl methacrylate (PMMA) generally have better mechanical properties and low immunogenicity. sex, but lacks biological function. Therefore, hydrogels composed of natural and synthetic polymers can satisfy in vivo applications.
对湿润和动态表面(包括生物组织)的粘附在很多领域都很重要，可以强烈粘合到生物组织的粘合剂将具有广泛的应用，从组织修复和药物递送。然而，现有的组织粘合剂远非理想，或具有细胞毒性、组织相容性差，或在湿润组织表面粘附力差。因此急需开发能在湿润组织表面具有良好粘合性和组织相容性的生物材料。Adhesion to wet and dynamic surfaces, including biological tissue, is important in many fields, and adhesives that can bond strongly to biological tissue will have a wide range of applications, ranging from tissue repair and drug delivery. However, existing tissue adhesives are far from ideal, either exhibiting cytotoxicity, poor tissue compatibility, or poor adhesion on wet tissue surfaces. Therefore, there is an urgent need to develop biomaterials with good adhesion and histocompatibility on wet tissue surfaces.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种粘性水凝胶材料、免缝合人工神经导管及其制备方法。粘性水凝胶材料可实现人工神经导管与湿润和动态组织表面的粘合，无需缝合，使用方便。In view of this, the object of the present invention is to provide a viscous hydrogel material, a suture-free artificial nerve guide and a preparation method thereof. The sticky hydrogel material can realize the adhesion of the artificial nerve guide to the wet and dynamic tissue surface without sutures and convenient use.
为了实现本发明的目的，本发明采用以下技术方案：In order to realize the purpose of the present invention, the present invention adopts following technical scheme:
一种粘性水凝胶材料的制备方法，包括：A method for preparing a viscous hydrogel material, comprising:
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；1) dissolving chitosan, sodium alginate and acrylamide in MES buffer to obtain a mixed solution;
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 evenly, then add N, N'-methylenebisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO4 were stirred at room temperature for 0.5-1 hour to obtain a viscous hydrogel material.
优选的，所述MES缓冲液的pH值为5。Preferably, the pH value of the MES buffer is 5.
优选的，所述混合溶液中各组分含量为：Preferably, the content of each component in the mixed solution is:
更优选的，所述混合溶液中各组分含量为：More preferably, the content of each component in the mixed solution is:
优选的，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。Preferably, the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride used in step (2) is 0.12 g per 1 mL of the mixed solution; N-hydroxy The dosage of succinimide is 0.12g per 1mL of the mixed solution; the dosage of N,N'-methylenebisacrylamide is 3.6μL per 1mL of the mixed solution, the concentration is 20g/ L of N, N'-methylenebisacrylamide; the amount of tetramethylethylenediamine added is 0.8 μL of tetramethylethylenediamine per 1 mL of the mixed solution; the amount of ammonium persulfate is 22.6 μL of 0.27M ammonium persulfate was added to 1 mL of the mixed solution; the dosage of CaSO4 was 19 μL of 0.75 M CaSO4 per 1 mL of the mixed solution.
本发明在制备粘性水凝胶时加入N，N'-亚甲基双丙烯酰胺作为交联剂，在四甲基乙二胺、过硫酸铵组成的氧化还原引发体系里，使丙烯酰胺发生自由基聚合反应，形成聚丙烯酰胺，海藻酸钠中活化的羟基可与聚丙烯酰胺形成酰胺键，可以增强壳聚糖水凝胶的力学性能，使其在制备过程中快速成型。除此之外，CaSO4中的钙离子与海藻酸钠溶液中的海藻酸发生离子耦合，形成交联网络，也可使粘性水晶胶材料在模具中快速成型。In the present invention, N,N'-methylenebisacrylamide is added as a cross-linking agent when preparing viscous hydrogel, and acrylamide can be freely generated in the redox initiation system composed of tetramethylethylenediamine and ammonium persulfate. The activated hydroxyl group in sodium alginate can form an amide bond with polyacrylamide, which can enhance the mechanical properties of chitosan hydrogel and make it rapid in the preparation process. In addition, the calcium ions in CaSO4 ionically couple with the alginic acid in the sodium alginate solution to form a cross-linked network, which also allows the viscous crystal glue material to be rapidly formed in the mold.
本发明还提供了一种粘性水凝胶材料，按照上述制备方法制得。The present invention also provides a viscous hydrogel material prepared according to the above preparation method.
该粘性水凝胶为多孔结构且无细胞毒性，有利于神经导管内外营养物质交换、细胞迁移以及组织再生，具有较强的组织相容性。The viscous hydrogel has a porous structure and no cytotoxicity, is beneficial to the exchange of nutrients inside and outside the nerve conduit, cell migration and tissue regeneration, and has strong tissue compatibility.
本发明还提供了一种免缝合人工神经导管，包括壳聚糖人工神经导管和分别粘合在所述壳聚糖人工神经导管两端端部的粘性水凝胶，所述粘性水凝胶由上文所述的粘性水凝胶材料成胶后形成。The present invention also provides a suture-free artificial nerve guide, comprising a chitosan artificial nerve guide and viscous hydrogels respectively bonded to two ends of the chitosan artificial nerve guide, and the viscous hydrogel consists of The cohesive hydrogel materials described above are formed after gelling.
本发明还提供了一种上述免缝合人工神经导管的制备方法，包括以下步骤：The present invention also provides a method for preparing the above-mentioned suture-free artificial nerve guide, comprising the following steps:
(1)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；(1) Putting the chitosan artificial nerve guide into the mould, leaving a vacant space of 1 to 2 cm in length at both ends of the mould;
(2)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。(2) The viscous hydrogel material is injected into the two ends of the chitosan artificial nerve conduit placed in the mold before being gelled, and placed for 12 to 24 hours until the viscous hydrogel material naturally forms After gluing, the mold is removed to avoid suturing the artificial nerve conduit.
优选的，所述模具与所述壳聚糖人工神经导管内外径匹配。Preferably, the mold matches the inner and outer diameters of the chitosan artificial nerve guide.
优选的，所述壳聚糖人工神经导管的长度为5mm。Preferably, the chitosan artificial nerve guide has a length of 5mm.
本发明还提供了一种免缝合人工神经导管，其特征在于，由权利要求1或2所述的粘性多孔水凝胶分别粘合在壳聚糖人工神经导管的两端制成。The present invention also provides a suture-free artificial nerve guide, which is characterized in that it is made by bonding the viscous porous hydrogel described in claim 1 or 2 to two ends of the chitosan artificial nerve guide respectively.
与现有技术相比，本发明公开的粘性水凝胶的制备方法中，将壳聚糖、海藻酸钠、丙烯酰胺混合后，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺混匀后再加入N，N'-亚甲基双丙烯酰胺、四甲基乙二胺、过硫酸铵和CaSO4，得到粘性水凝胶材料，该粘性水凝胶为多孔结构，且无细胞毒性，生物相容性好，适合植入体内。另外，粘性水凝胶材料制备过程中加入的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺使海藻酸钠中的羧基活化后，可与壳聚糖以及人体组织中氨基，形成酰胺键，形成交联网络。因此，将该粘性水凝胶材料在成胶前注射到放置有壳聚糖人工神经导管的模具中的两端位置，海藻酸钠中的羟基通过与壳聚糖形成酰胺键，在壳聚糖神经导管端部位置强力粘合成型，制备形成免缝合人工神经导管。该免缝合人工神经导管在使用过程中，譬如神经导管手术植入时，使粘性水凝胶能够通过与神经组织中的氨基形成酰胺键从而强力粘附神经组织，达到免去手术缝合的效果，使用方便。Compared with the prior art, in the preparation method of the viscous hydrogel disclosed by the present invention, after mixing chitosan, sodium alginate and acrylamide, 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride, N-hydroxysuccinimide and N, N'-methylenebisacrylamide, tetramethylethylenediamine, ammonium persulfate and CaSO4 are added to obtain viscous water A gel material, the viscous hydrogel has a porous structure, has no cytotoxicity, has good biocompatibility, and is suitable for implantation in the body. In addition, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide added during the preparation of viscous hydrogel materials make the carboxyl groups in sodium alginate After activation, it can form amide bonds with chitosan and amino groups in human tissues to form a cross-linked network. Therefore, the viscous hydrogel material is injected into the two ends of the mold with the chitosan artificial nerve guide before gelling, the hydroxyl group in the sodium alginate forms an amide bond with the chitosan, and the The end of the nerve guide is strongly bonded and formed to form a suture-free artificial nerve guide. During the use of the suture-free artificial nerve guide, such as when the nerve guide is surgically implanted, the viscous hydrogel can form an amide bond with the amino group in the nerve tissue to strongly adhere to the nerve tissue, achieving the effect of eliminating surgical sutures. Easy to use.
具体实施方式Detailed ways
为了进一步理解本发明，下面结合实施例对本发明优选实施方案进行描述，但是应当理解，这些描述只是为了进一步说明本发明的特征和优点，而不是对本发明权利要求的限制。In order to further understand the present invention, the preferred embodiments of the present invention are described below in conjunction with the examples, but it should be understood that these descriptions are only to further illustrate the features and advantages of the present invention, rather than limiting the claims of the present invention.
本发明所有原料，对其来源没有特别限制，在市场上购买的或按照本领域技术人员熟知的常规方法制备的即可。All raw materials in the present invention have no particular limitation on their sources, they can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.
实施例1Example 1
1)配制1L的MES缓冲液，调节pH至5。1) Prepare 1L of MES buffer and adjust the pH to 5.
2)称取20g壳聚糖、20g海藻酸钠和120g丙烯酰胺，溶于配置得到的MES缓冲液中，得到混合溶液。2) Weighing 20g of chitosan, 20g of sodium alginate and 120g of acrylamide, and dissolving them in the prepared MES buffer to obtain a mixed solution.
3)向混合溶液中加入120g氨基交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、120g的N-羟基琥珀酰亚胺(NHS)，混匀后加入3.6mL的浓度为20g/L的共价交联剂N，N'-亚甲基双丙烯酰胺(MBAA)，0.8mL的加速剂四甲基乙二胺(TEMED)、22.6mL的浓度为0.27M引发剂过硫酸铵(APS)和19.1mL的浓度为0.75M离子交联剂CaSO4，混合均匀，得到粘性水凝胶材料。3) Add 120g of amino crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 120g of N-hydroxysuccinimide (NHS ), after mixing, add 3.6 mL of covalent cross-linking agent N, N'-methylenebisacrylamide (MBAA) with a concentration of 20 g/L, 0.8 mL of accelerator tetramethylethylenediamine (TEMED), 22.6 mL of 0.27M initiator ammonium persulfate (APS) and 19.1 mL of 0.75M ion crosslinker CaSO4 were mixed uniformly to obtain a viscous hydrogel material.
5)将壳聚糖人工神经导管放入内外径匹配的塑料模具，两端留出2cm空余。5) Put the chitosan artificial nerve guide into a plastic mold with matching inner and outer diameters, leaving 2 cm free at both ends.
6)在粘性水凝胶材料未成胶之前将其注射到壳聚糖导管两端空余空间，放置12h，自然成胶。6) Before the viscous hydrogel material is gelled, it is injected into the empty space at both ends of the chitosan catheter, left for 12 hours, and gelled naturally.
7)卸掉模具，放入蒸馏水内泡洗，去除未交联的交联剂和丙烯酰胺。7) Remove the mold, soak it in distilled water to remove uncrosslinked crosslinking agent and acrylamide.
实施例2Example 2
1)配制1L的MES缓冲液，调节pH至5。1) Prepare 1L of MES buffer and adjust the pH to 5.
2)称取30g壳聚糖、30g海藻酸钠和120g丙烯酰胺，溶于配置得到的MES缓冲液中，得到混合溶液。2) Weigh 30g of chitosan, 30g of sodium alginate and 120g of acrylamide, and dissolve them in the prepared MES buffer to obtain a mixed solution.
3)向混合溶液中加入120g氨基交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、120g的N-羟基琥珀酰亚胺(NHS)，混匀后加入3.6mL的浓度为20g/L的共价交联剂N，N'-亚甲基双丙烯酰胺(MBAA)，0.8mL的加速剂四甲基乙二胺(TEMED)、22.6mL的浓度为0.27M引发剂过硫酸铵(APS)和19.1mL的浓度为0.75M离子交联剂CaSO4，混合均匀，得到粘性水凝胶材料。3) Add 120g of amino crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 120g of N-hydroxysuccinimide (NHS ), after mixing, add 3.6 mL of covalent cross-linking agent N, N'-methylenebisacrylamide (MBAA) with a concentration of 20 g/L, 0.8 mL of accelerator tetramethylethylenediamine (TEMED), 22.6 mL of 0.27M initiator ammonium persulfate (APS) and 19.1 mL of 0.75M ion crosslinker CaSO4 were mixed uniformly to obtain a viscous hydrogel material.
5)将壳聚糖人工神经导管放入内外径匹配的塑料模具，两端留出2cm空余。5) Put the chitosan artificial nerve guide into a plastic mold with matching inner and outer diameters, leaving 2 cm free at both ends.
6)在水凝胶未成胶之前将其注射到壳聚糖导管两端空余空间，放置18h，自然成胶。6) Inject the hydrogel into the empty space at both ends of the chitosan catheter before it is gelled, and let it stand for 18 hours to form a gel naturally.
7)卸掉模具，放入蒸馏水内泡洗，去除未交联的交联剂和丙烯酰胺。7) Remove the mold, soak it in distilled water to remove uncrosslinked crosslinking agent and acrylamide.
实施例3Example 3
1)配制1L的MES缓冲液，调节pH至5。1) Prepare 1L of MES buffer and adjust the pH to 5.
2)称取10g壳聚糖、10g海藻酸钠和70g丙烯酰胺，溶于配置得到的MES缓冲液中，得到混合溶液。2) Weighing 10 g of chitosan, 10 g of sodium alginate and 70 g of acrylamide, and dissolving them in the prepared MES buffer to obtain a mixed solution.
3)向混合溶液中加入120g氨基交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、120g的N-羟基琥珀酰亚胺(NHS)，混匀后加入3.6mL的浓度为20g/L的共价交联剂N，N'-亚甲基双丙烯酰胺(MBAA)，0.8mL的加速剂四甲基乙二胺(TEMED)、22.6mL的浓度为0.27M引发剂过硫酸铵(APS)和19.1mL的浓度为0.75M离子交联剂CaSO4，混合均匀，得到粘性水凝胶材料。3) Add 120g of amino crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 120g of N-hydroxysuccinimide (NHS ), after mixing, add 3.6 mL of covalent cross-linking agent N, N'-methylenebisacrylamide (MBAA) with a concentration of 20 g/L, 0.8 mL of accelerator tetramethylethylenediamine (TEMED), 22.6 mL of 0.27M initiator ammonium persulfate (APS) and 19.1 mL of 0.75M ion crosslinker CaSO4 were mixed uniformly to obtain a viscous hydrogel material.
5)将壳聚糖人工神经导管放入内外径匹配的塑料模具，两端留出2cm空余。5) Put the chitosan artificial nerve guide into a plastic mold with matching inner and outer diameters, leaving 2 cm free at both ends.
6)在水凝胶未成胶之前将其注射到壳聚糖导管两端空余空间，放置24h，自然成胶。6) Inject the hydrogel into the empty space at both ends of the chitosan catheter before it is gelled, leave it for 24 hours, and gel it naturally.
7)卸掉模具，放入蒸馏水内泡洗，去除未交联的交联剂和丙烯酰胺。7) Remove the mold, soak it in distilled water to remove uncrosslinked crosslinking agent and acrylamide.
对比例1Comparative example 1
1)配制1L的MES缓冲液，调节pH至5。1) Prepare 1L of MES buffer and adjust the pH to 5.
2)称取20g壳聚糖、20g海藻酸钠和120g丙烯酰胺，溶于配置得到的MES缓冲液中，得到混合溶液。2) Weighing 20g of chitosan, 20g of sodium alginate and 120g of acrylamide, and dissolving them in the prepared MES buffer to obtain a mixed solution.
3)向混合溶液中加入120g氨基交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、120g的N-羟基琥珀酰亚胺(NHS)，混匀后加入0.8mL的加速剂四甲基乙二胺(TEMED)、22.6mL的浓度为0.27M引发剂过硫酸铵(APS)和19.1mL的浓度为0.75M离子交联剂CaSO4，混合均匀，得到粘性水凝胶材料。3) Add 120g of amino crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 120g of N-hydroxysuccinimide (NHS ), after mixing, add 0.8mL of accelerator tetramethylethylenediamine (TEMED), 22.6mL of 0.27M initiator ammonium persulfate (APS) and 19.1mL of 0.75M ion crosslinker CaSO4 , mixed evenly to obtain a viscous hydrogel material.
5)将壳聚糖人工神经导管放入内外径匹配的塑料模具，两端留出2cm空余。5) Put the chitosan artificial nerve guide into a plastic mold with matching inner and outer diameters, leaving 2 cm free at both ends.
6)在粘性水凝胶材料未成胶之前将其注射到壳聚糖导管两端空余空间，放置18h，自然成胶。6) Before the viscous hydrogel material is gelled, it is injected into the empty space at both ends of the chitosan catheter, left for 18 hours, and gelled naturally.
7)卸掉模具，放入蒸馏水内泡洗，去除未交联的交联剂和丙烯酰胺。7) Remove the mold, soak it in distilled water to remove uncrosslinked crosslinking agent and acrylamide.
对比例2Comparative example 2
1)配制1L的MES缓冲液，调节pH至5。1) Prepare 1L of MES buffer and adjust the pH to 5.
2)称取20g壳聚糖、20g海藻酸钠和120g丙烯酰胺，溶于配置得到的MES缓冲液中，得到混合溶液。2) Weighing 20g of chitosan, 20g of sodium alginate and 120g of acrylamide, and dissolving them in the prepared MES buffer to obtain a mixed solution.
3)向混合溶液中加入120g氨基交联剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)、120g的N-羟基琥珀酰亚胺(NHS)，混匀后加入3.6mL的浓度为20g/L的共价交联剂N，N'-亚甲基双丙烯酰胺(MBAA)、0.8mL的加速剂四甲基乙二胺(TEMED)和19.1mL的浓度为0.75M离子交联剂CaSO4，混合均匀，得到粘性水凝胶材料。3) Add 120g of amino crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 120g of N-hydroxysuccinimide (NHS ), after mixing, add 3.6 mL of covalent cross-linking agent N, N'-methylenebisacrylamide (MBAA) with a concentration of 20 g/L, 0.8 mL of accelerator tetramethylethylenediamine (TEMED) and 19.1 mL of 0.75 M ionic crosslinker CaSO4 was mixed uniformly to obtain a viscous hydrogel material.
5)将壳聚糖人工神经导管放入内外径匹配的塑料模具，两端留出2cm空余。5) Put the chitosan artificial nerve guide into a plastic mold with matching inner and outer diameters, leaving 2 cm free at both ends.
6)在粘性水凝胶材料未成胶之前将其注射到壳聚糖导管两端空余空间，放置40h，自然成胶。6) Before the viscous hydrogel material is gelled, it is injected into the empty space at both ends of the chitosan catheter, left for 40 hours, and gelled naturally.
7)卸掉模具，放入蒸馏水内泡洗，去除未交联的交联剂和丙烯酰胺。7) Remove the mold, soak it in distilled water to remove uncrosslinked crosslinking agent and acrylamide.
测试例：粘性水凝胶粘合力测试Test Example: Viscous Hydrogel Adhesion Test
将大鼠坐骨神经塞入本发明实施例1-3制备得到的免缝合人工神经导管两端的粘性水凝胶中1mm处，利用手术钳在37℃潮湿条件下固定10min。使用万能力学测试机(TFW-58,Shanghai Tuofeng Instrument Technology Co.Ltd.,China).以0.5mm/min的十字头速度进行测试，测试结果如表1所示。The rat sciatic nerve was stuffed into 1 mm of the viscous hydrogel at both ends of the suture-free artificial nerve guide prepared in Example 1-3 of the present invention, and fixed with surgical forceps for 10 min under humid conditions at 37°C. A universal mechanical testing machine (TFW-58, Shanghai Tuofeng Instrument Technology Co. Ltd., China) was used to test at a crosshead speed of 0.5 mm/min. The test results are shown in Table 1.
表1人工神经导管对神经的粘附力Table 1 Adhesion force of artificial nerve guide to nerve
根据表1测试结果可以发现，本发明实施例和对比例所提供的免缝合人工神经导管两端的粘性水凝胶对神经导管的粘附力在0.01～0.1MPa之间，其中实施例1(浓度为壳聚糖，20g/L；海藻酸钠，20g/L；丙烯酰胺，120g/L)制备得到的免缝合人工神经导管两端的粘性水凝胶对神经粘附力最强，可达到0.1MPa。另外，根据对比例可以发现去除四甲基乙二胺(TEMED)会导致粘性多孔水凝胶粘合力下降，去除过硫酸铵(APS)基本不会使粘性水凝胶的粘合力下降，但会导致成胶时间延长。According to the test results in Table 1, it can be found that the adhesion of the adhesive hydrogel at both ends of the suture-free artificial nerve guide provided by the embodiments of the present invention and the comparative examples to the nerve guide is between 0.01 and 0.1 MPa, where in Example 1 (concentration Chitosan, 20g/L; sodium alginate, 20g/L; acrylamide, 120g/L) The adhesive hydrogel at both ends of the suture-free artificial nerve guide has the strongest adhesion to the nerve, which can reach 0.1MPa . In addition, according to the comparative examples, it can be found that removing tetramethylethylenediamine (TEMED) will cause the adhesive force of the viscous porous hydrogel to decrease, and removing ammonium persulfate (APS) will not substantially reduce the adhesive force of the viscous hydrogel, But it will lead to prolonged gelation time.
以上显示和描述了本发明的基本原理和主要特征和本发明的优点，对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。The basic principles and main features of the present invention and the advantages of the present invention have been shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-mentioned exemplary embodiments, and without departing from the spirit or basic principles of the present invention. The present invention can be implemented in other specific forms without any specific features. Accordingly, the embodiments should be regarded in all points of view as exemplary and not restrictive, the scope of the invention being defined by the appended claims rather than the foregoing description, and it is therefore intended that the scope of the invention be defined by the appended claims rather than by the foregoing description. All changes within the meaning and range of equivalents of the elements are embraced in the present invention. Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.
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| CN201910874185.1ACN110448727B (en) | 2019-09-17 | 2019-09-17 | A kind of viscous hydrogel material, suture-free artificial nerve conduit and preparation method thereof |
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| CN201910874185.1ACN110448727B (en) | 2019-09-17 | 2019-09-17 | A kind of viscous hydrogel material, suture-free artificial nerve conduit and preparation method thereof |
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