WO2022100276A1 - 一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法 - Google Patents
一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法 Download PDFInfo
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Definitions
- the application belongs to the technical field of biomedical materials, and in particular relates to a composite hydrogel of a directional fibrin and polypeptide interpenetrating network for nerve regeneration and a preparation method thereof.
- Nerve injury is a common clinical problem in the world, including central nerve injury and peripheral nerve injury. It has the characteristics of high morbidity, high disability rate and younger age. Nerve injury seriously affects the normal life of patients and family happiness, and also brings a huge economic burden to the society. At present, there is no effective and convenient treatment and method for nerve injury repair.
- the repair after nerve injury is a series of complex pathological processes. The slow regeneration of nerves, the formation of scar tissue, the inhibition of the generation of microenvironment, motor terminal tissue, functional degradation and other factors limit the repair of injured nerves.
- tissue engineering technology has been widely valued. Among them, the construction of tissue-engineered neural scaffolds is a research hotspot and an effective method in the treatment of nerve regeneration.
- Nerve conduits are tubular structures that are sutured or fixed at both ends of the nerve defect to provide space for nerve regeneration. They can guide axon regeneration and prevent the damaged part from being invaded by scar tissue and cells. Blocks the process of nerve regeneration.
- the simple nerve conduit itself does not have many factors that regulate nerve repair, such as neurotrophic factors and nerve orientation structure, resulting in poor repair effect; in addition, long-distance and large-scale nerve defects are difficult to repair with simple nerve conduits.
- a neural scaffold material with multi-function and multi-signal which can support the distribution of cells and the growth of regenerated axons in three-dimensional space while providing space for nerve ingrowth, and at the same time ensure the cell affinity of the material.
- the properties and mechanical properties can meet the needs of regeneration and repair of nerve injury.
- Fibrin is a widely used traditional biological material, which is formed by the polymerization of soluble precursor molecule fibrinogen under the action of thrombin. Fibrin plays an important role in hemostasis and damaged tissue repair, not only as a scaffold material, but also in regulating the behavior of damaged cells.
- Chinese patent CN201611258868.7 discloses a biomimetic fibrin hydrogel bundle for spinal cord injury repair and a preparation method thereof. It is prepared from fibrin by electrospinning technology, which can be compounded with one or more polysaccharides and has a multi-level orientation structure.
- single fibrin or complex polysaccharide hydrogels lack growth factors that promote tissue regeneration, and have limited repair effects on long-distance and large-sized damaged nerves.
- the fibrin molecule has a large number of binding sites for growth factors, integrins and other ECM components, which can be modified to increase signals beneficial to nerve regeneration and achieve better repair effects.
- Growth factors are a class of small molecular proteins or polypeptides that have biological effects such as stimulating cell proliferation and inducing cell differentiation.
- the presence of local growth factors at the site of injury plays a crucial role in modulating phenotypic changes in various neural and non-neural cells.
- neurotrophic factors are extremely deficient in the pathological environment.
- endogenous growth factors secreted by nerve cells at the distal nerve stump can support axon regeneration to a certain extent, the efficiency and persistence of the factors secreted by such cells are not sufficient. time is limited. Therefore, nerve injury repair needs to rely on the addition of exogenous growth factors.
- exogenous growth factors there are still many problems in the clinical application of exogenous growth factors.
- bioactive polypeptide fragments that mimic the function of growth factors have the advantages of stable physicochemical properties, long-lasting activity, non-toxic, harmless, and degradable, and are widely used as substitutes for growth factors.
- the present application proposes a directional fibrin-polypeptide interpenetrating network composite hydrogel for nerve regeneration.
- the composite hydrogel has a multi-level orientation structure, the diameter of the composite hydrogel is 0.5-5.0mm, and the composite hydrogel is composed of micron fiber tows, and the diameter of the microfiber tow is 10 ⁇ m-100 ⁇ m;
- the tow is composed of nanofiber tow, and the diameter of the nanofiber tow is 100nm-500nm.
- polypeptides are compounded in the composite hydrogel, and the polypeptides are self-assembling polypeptides; further, the self-assembling polypeptides include functional polypeptides; further, the functional polypeptides include artificially synthesized laminar adhesions Self-assembling polypeptides grafted with functionally active short peptides of protein, fibronectin, cadherin, nerve growth factor, brain-derived nerve growth factor or vascular endothelial growth factor.
- the content of functional polypeptide in the polypeptide is 10wt%-100wt%.
- a preparation method of a directional fibrin and polypeptide interpenetrating network composite hydrogel for nerve regeneration comprising the following steps:
- the CaCl 2 aqueous solution is mixed with the thrombin solution to obtain a cross-linked solution and incubated at 37° C.
- the mass fraction of CaCl 2 in the cross-linked solution is 0.5%-3%, and the thrombin content is 4-50 Units/mL.
- the cross-linking solution is placed in a rotating receiving tray for use;
- the preparation method of the electrospinning stock solution in the step 2) is as follows: the fibrinogen solution and the polypeptide solution are mixed evenly, and then the polyethylene oxide aqueous solution is added and mixed evenly to obtain the electrospinning stock solution.
- the preparation method of the electrospinning stock solution in the step 2) is as follows: the fibrinogen solution and the polyethylene oxide aqueous solution are mixed uniformly to obtain the electrospinning stock solution; after step 3), the following steps are also included: soak the composite product in the polypeptide solution. medium, and let stand for 20 min after equilibration.
- the preparation method of the fibrinogen solution is as follows: dissolving the fibrinogen in pure water to obtain a fibrinogen solution, wherein the mass fraction of the fibrinogen is 2%-8%;
- the preparation method of the polyoxyethylene aqueous solution is as follows: dissolving the polyoxyethylene in pure water to obtain the polyoxyethylene aqueous solution, wherein the mass fraction of the polyoxyethylene is 0.1%-0.8%;
- the preparation method of the polypeptide solution is as follows: the functional polypeptide powder is dissolved in pure water, and the polypeptide solution is obtained after dispersion;
- the cross-linking reaction is formed by self-assembly and cross-linking of polypeptides.
- the cross-linking reaction includes an enzymatic cross-linking method in which glutamine transaminase catalyzes an acyl transfer reaction, and EDC/NHS catalyzes chemical cross-linking of fibrin and polypeptide fragments to form amide bonds. Methods, using glutaraldehyde for chemical cross-linking and genipin for chemical cross-linking.
- the conditions of the step 4) cross-linking reaction are: 37°C temperature-controlled cross-linking, phosphate buffer solution cross-linking, 10-30Units/mL glutamine transaminase phosphate buffer solution cross-linking, and the concentration is 10-60mmol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide+concentration is 3-30mmol/L N-hydroxysuccinimide+concentration is 50mmol/L potassium dihydrogen phosphate solution One or more of them are used in combination.
- the composite hydrogel of oriented fibrin and functional polypeptide prepared by single-channel electrospinning technology in this application is a hydrogel material with high water content, which is composed of multi-level oriented nanofiber bundles with highly simulated nerve tissue. , and more importantly, functional polypeptide fragments with biochemical activity that simulate the function of growth factors at the same time.
- the directional fibrin and functional polypeptide composite hydrogels exhibit similar compositional structure, mechanical properties, extracellular matrix environment to natural nerve tissue. And bioactive factors, it has excellent functions of promoting nerve cell adhesion, directional growth of nerve fibers, inducing nerve and angiogenesis repair, and can be applied to nerve regeneration and repair of brain, spinal cord and peripheral nerve injury.
- the present application can overcome the deficiencies of the biological activity of the existing catheter materials and filler materials used for nerve damage repair, and the problems of poor safety and low cost performance of the current growth factor use, and provide a directional fibrin and functional polypeptide composite hydrogel, It can be used for the repair and treatment of nerve tissue damage. It is a new type of composite material for nerve tissue defect repair.
- the composite hydrogel has a microstructure that induces the directional growth of nerves at the defect, and at the same time realizes the controlled release of functional polypeptide fragments, plays a role similar to growth factors, and promotes the regeneration of nerve tissue and surrounding blood vessels, while avoiding the disorder of the regenerated axons. Unpredictable and unnecessary physiological or pathological effects of growth and the addition of exogenous growth factors.
- the present application constructs a functionally oriented fiber composite hydrogel with neuron biomimetic directional structure and nerve-like growth factors that promotes nerve repair.
- the composite hydrogel improves the growth environment of damaged nerves in terms of structure and composition, and is beneficial to the repair of nerve cells and the generation of directional axons.
- This application combines the physical properties of directional structures for nerve repair and the biochemical properties of growth factors for nerve repair to prepare directional functional nanofiber hydrogels to make up for the structural and functional deficiencies of existing nerve repair materials.
- a composite nerve substitute with promising application in promoting nerve regeneration.
- the oriented fibrin claimed in this application is prepared by electrospinning technology received in a liquid state, and the obtained multi-level oriented structure with micro-nano scale is intended to simulate the natural oriented structure of nerve fibers and the way that nerve fibers pass through. Endogenous structures assembled at the endoneurium, perineurium, and epineurium levels. Different from the previous non-oriented fibrin hydrogel, the oriented fibrin hydrogel of the present application is a biomimetic hydrogel material with a unique biomimetic design structure.
- the present application is different from the simple self-assembled polypeptide hydrogel.
- the function of the polypeptide is combined with the directional fibrin hydrogel through the interpenetrating network. Therefore, the composite hydrogel material is formed by the functional oriented nanofibers oriented step by step, and finally presents a functional fibrin hydrogel with a multi-level oriented structure.
- the composite hydrogel material of the present application fully combines the characteristics of oriented fibrin hydrogel and self-assembled polypeptide hydrogel to form a biomimetic oriented hydrogel material with specific induction function of regeneration and repair. Different from the above two materials alone, the composite hydrogel material of the present application forms new nano-functional fibers, and the hydrogel is composed of new functional nano-fibers.
- the composite nanohydrogels are different from individual directional fibrin and self-assembled functional polypeptides; in the experimental models of peripheral nerve and spinal cord nerve repair, the composite nanohydrogels
- the oriented fibrin hydrogel is also significantly better than the simple fibrin and functional polypeptide group, and has a more excellent function of promoting nerve regeneration and repair.
- Fig. 1 is the physical photograph of composite hydrogel
- Fig. 2 is the scanning electron microscope picture of composite hydrogel
- Figure 3 is a cross-sectional transmission electron microscope image of the composite hydrogel
- Figure 4 is a graph showing the results of cell staining 3 days after the hydrogel was inoculated with Schwann cells
- Figure 5 is a photo of the operation of the composite hydrogel implanted in the animal sciatic nerve defect
- Figure 6 is a graph showing the results of toluidine blue staining 12 weeks after the composite hydrogel was transplanted into the sciatic nerve defect of an animal;
- Figure 7 is a photo of the operation of the composite hydrogel transplanted on the half-transected defect of the animal spinal cord;
- Figure 8 is a graph showing the results of hematoxylin-eosin staining 12 weeks after the composite hydrogel was transplanted into the spinal cord hemi-transection defect.
- the present application provides an oriented fibrin and polypeptide interpenetrating network composite hydrogel for nerve regeneration and a preparation method thereof.
- the oriented fibrin and polypeptide interpenetrating network composite hydrogel combines molecular self-assembly by electrospinning technology , enzymatic cross-linking or physical and chemical cross-linking.
- the design idea of the oriented fibrin and functional polypeptide interpenetrating network composite hydrogel designed in this application is based on the special structure and properties of natural neural tissue.
- the hydrogel has a multi-level oriented structure, and the fiber size ranges from nanometer to micrometer.
- the structure is composed of nanofiber tows, the nanofiber tows first form micro-fiber tows, and then the micro-fiber tows form macroscopic hydrogel bundles.
- the diameter of the nanofiber tow is 100nm-500nm, and the diameter of the microfiber tow is 10 ⁇ m-100 ⁇ m.
- the diameter of the finally prepared oriented fibrin-polypeptide interpenetrating network composite hydrogel is 0.5-5.0 mm.
- the oriented fibrin and polypeptide interpenetrating network composite hydrogel of the present application is a hydrogel material with high water content, its composition, structure and mechanical properties are highly biomimetic to the extracellular matrix of natural neural tissue, and has the function of simulating growth factors
- the polypeptide fragment has excellent effect of promoting the directional growth of nerve fibers and inducing nerve and angiogenesis.
- the composite hydrogel exhibits the appearance of oriented fiber bundles with uniform diameters.
- the fibrin fibers were neatly arranged under a scanning electron microscope, showing the characteristics of multi-level orientation; and the self-assembled polypeptide nanofiber tow was attached to the surface of the microfiber tow, which is the fibrin Nanofiber tows add multiple surface microstructures.
- the Schwann cells exhibited a directional arrangement on the composite hydrogel, which was consistent with the orientation of the composite hydrogel itself, indicating that the composite hydrogel could induce the directional growth of Schwann cells.
- the composite hydrogel material of the present application was transplanted into the transection defect of the T9 segment of the spinal cord of the rat as shown in Figure 7. After 12 weeks, the spinal cord nerve tissue could regenerate in the defect.
- Figure 8 shows the tissue staining results of the injury site.
- the specific diameter and length of the oriented fibrin and functional polypeptide composite hydrogel can be determined according to the diameter and length of the defected nerve required for surgery.
- the preparation method of the directional fibrin and functional polypeptide interpenetrating network composite hydrogel for nerve regeneration in the present application includes:
- the above-mentioned physically combined oriented fibrin and functional polypeptide complexes are placed in cross-linking conditions for in situ cross-linking to obtain the oriented fiber hydrogel of the cross-linked fibrin complex functional polypeptides, and the above-mentioned cross-linking conditions include one of the following or Several: 37°C temperature-controlled cross-linking, phosphate buffered saline (PBS) solution cross-linking, 10-30Units/mL transglutaminase in PBS solution cross-linking and 10-60mmol/L 1-ethyl-3-(3- Dimethylaminopropyl) carbodiimide (EDC) + 3-30 mmol/LN-hydroxysuccinimide (NHS) + 50 mmol/L potassium dihydrogen phosphate solution for cross-linking, etc.
- PBS phosphate buffered saline
- EDC 1-ethyl-3-(3- Dimethylaminopropyl) carbodiimide
- the cross-linking conditions include one or more of the following: temperature-controlled cross-linking at 37° C., phosphate buffer solution (PBS) solution cross-linking , 10-30Units/mL transglutaminase in PBS solution cross-linking and 10-60mmol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)+3-30mmol/LN -Hydroxysuccinimide (NHS)+50mmol/L potassium dihydrogen phosphate solution for cross-linking, etc.
- PBS phosphate buffer solution
- EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
- NHS 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
- One or more functional polypeptides are compounded in the composite fiber hydrogel, and the functional polypeptides are artificially synthesized laminin (IKVAV, the same as the abbreviation for the amino acid sequence), fibronectin (RGD, the same as the abbreviation for the amino acid sequence), cadherin (HAVDI) , the amino acid sequence is the same as the abbreviation), nerve growth factor (CTD, CTDIKGKCTGACDGKQC), brain-derived nerve growth factor (RGI, RGIDKRHWNSQ), vascular endothelial growth factor (KLT, KLTWQELYQLKYKGI) functionally active short peptide grafted self-assembling polypeptide (RADA). , RADARADARADARADA), self-assembling peptide mixture.
- the functional polypeptide fragments and self-assembling polypeptide fragments are not limited thereto.
- the artificially synthesized self-assembled polypeptide RADA powders grafted with the functional polypeptides of IKVAV and CTD were dissolved in pure water, respectively, and after ultrasonic dispersion, the self-assembled polypeptide solution with the function of brain-derived nerve growth factor and the blood vessel were obtained respectively.
- Endothelial growth factor functional self-assembled polypeptide solution, the mass fraction of the two functional self-assembled polypeptide solutions and the self-assembled polypeptide solution are both 1%, the content of the above two functional self-assembled polypeptides are 25% and 50% respectively, and the self-assembled polypeptide solution The content is 25%, and the obtained bifunctional self-assembled polypeptide solution is placed in a 4° C. refrigerator for use.
- the fibrin aqueous solution is mixed with the two functional self-assembled polypeptide solutions, the concentration of fibrinogen is 2%, the mass fraction of the two polypeptide solutions is 1%, and the content of the above two self-assembled polypeptides is both 50%; mixing;
- the solution is mixed with polyethylene oxide aqueous solution, the concentration is 0.5%, and the above solution is injected into a syringe for use.
- the electrospinning stock solution injected into the syringe was electrospun under the action of the propelling pump.
- the bolus injection rate of the fibrinogen solution was adjusted to 2.5 mL/h, and the loading voltage was 4 kV to obtain continuous and stable fibrinogen spinning.
- the fibrinogen spinning obtained by electrospinning was received by a metal selective turntable, and the rotation receiving speed was adjusted to 50 rpm, and the composite fibrin spinning was under the action of the cross-linking solution to obtain a directional bifunctional fibrin hydrogel. Oriented fibrin hydrogels with a diameter of 1 mm were collected.
- the fibrin aqueous solution is mixed with the two functional self-assembled polypeptide solutions, the concentration of fibrinogen is 2%, the mass fraction of the two polypeptide solutions is 1%, and the content of the above two functional self-assembled polypeptides is 25% and 25%, respectively. 50%, the content of the self-assembled polypeptide solution is 25%; the mixed solution is mixed with polyethylene oxide aqueous solution, the concentration is 0.5%, and the above solution is injected into a syringe for use.
- the electrospinning stock solution injected into the syringe was electrospun under the action of the propelling pump.
- the bolus injection rate of the fibrinogen solution was adjusted to 2.5 mL/h, and the loading voltage was 4 kV to obtain continuous and stable fibrinogen spinning.
- the fibrinogen spinning obtained by electrospinning was received by a metal selective turntable, and the rotation receiving speed was adjusted to 50 rpm, and the composite fibrin spinning obtained directional bifunctional fibrin hydrogel under the action of the cross-linking solution. Oriented fibrin hydrogels with a diameter of 1 mm were collected.
- the fibrin aqueous solution is mixed with the functional self-assembled polypeptide solution, the concentration of fibrinogen is 2%, the mass fraction of the polypeptide solution is 1%, the content of the above-mentioned functional self-assembled polypeptide is 50%, and the content of the self-assembled polypeptide solution is 50% ;
- the mixed solution is mixed with polyethylene oxide aqueous solution, the concentration is 0.5%, and the above solution is injected into the syringe for use.
- the electrospinning stock solution injected into the syringe was electrospun under the action of the propelling pump.
- the bolus injection rate of the fibrinogen solution was adjusted to 2.5 mL/h, and the loading voltage was 4 kV to obtain continuous and stable fibrinogen spinning.
- the fibrinogen spinning obtained by electrospinning was received by a metal selective turntable, and the rotation receiving speed was adjusted to 50 rpm, and the composite fibrin spinning was under the action of the cross-linking solution to obtain a directional functional fibrin hydrogel. Oriented fibrin hydrogels with a diameter of 1 mm were collected.
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Abstract
本申请公开了一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法。所述复合水凝胶是以纤维蛋白和多肽互穿的复合纳米纤维结构组成。通过静电纺丝制备定向的纤维蛋白水凝胶，交联多肽通过溶胀及物理吸附与纤维蛋白进行互穿，再经过多种交联方式使其稳定结合，形成定向的互穿网络复合水凝胶。本申请复合水凝胶是一种高含水量的复合水凝胶，其组成、结构、力学性能高度仿生天然神经组织的细胞外基质，含有模拟生长因子功能的多肽片段，具有优异的促进神经细胞黏附、神经纤维定向生长，诱导神经和血管再生的作用，可应用于脑、脊髓和外周神经损伤的神经再生修复。
Description
本申请要求于2020年11月16日提交中国专利局、申请号为202011280197.0、申请名称为“一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
本申请属于生物医用材料技术领域，尤其涉及一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法。
神经损伤是世界上常见的临床难题，包括中枢神经损伤和周围神经损伤，具有高发病率、高致残率和低龄化的特点，是医学界亟待解决的重大问题。神经损伤严重影响了患者的正常生活和家庭幸福，还会给社会带来巨大的经济负担，目前尚无有效且便捷的治疗手段和方法用于神经损伤修复。神经损伤后的修复是一系列复杂的病理过程，神经再生过程缓慢，疤痕组织的形成，抑制微环境的产生，运动终端组织、功能退化等因素限制了损伤神经的修复。针对目前临床上遇到的困难，组织工程技术被广泛重视。其中，构建组织工程神经支架是神经再生治疗方法中的研究热点和有效方法。目前相关的神经桥接材料多为神经导管，神经导管是缝合或固定在神经缺损两端，为神经再生提供空间的管状结构，它能够引导轴突再生，防止损伤部分受到瘢痕组织和细胞的侵袭而阻碍神经再生过程。然而，简单的神经导管本身不具有神经营养因子和神经的取向结构等众多调控神经修复的因素，导致修复效果不佳；此外，长距离、大尺寸的神经缺损难以通过简单的神经导管来修复。因此，有必要开发一种具备多功能多信号的神经支架材料，能够在提供神经长入空间的同时，在三维空间内支持细胞的分布和再生轴突的生长，同时能够保障材料的细胞亲和性、力学性能等满足神经损伤的再生修复需求。
纤维蛋白是一种广泛应用的传统生物材料，在凝血酶的作用下由可溶性的前体分子纤维蛋白原聚合形成。纤维蛋白在止血和受损组织修复中起着重要作用，不仅可以作为支架材料，还能调控受损后细胞的行为。中国专利CN201611258868.7公开了一种用于脊髓损伤修复的仿生纤维蛋白水凝胶束及其制备方法。它是由纤维蛋白通过静电纺丝技术制备而成，可以复合一种或多种多糖，具有多级取向性结构。但是，单一的纤维蛋白或复合多糖水凝胶缺少促进组织再生的生长因子等，对长距离、大尺寸的损伤神经的修复效果有限。纤维蛋白分子上有大量的生长因子、整联蛋白以及其他ECM成分的结合位点，能够通过改性来增加有利于神经 再生的信号，达到更好的修复效果。
生长因子是一类具有刺激细胞增殖、诱导细胞分化等生物学效应的小分子蛋白或多肽，对诱导细胞增殖分化、组织或器官的修复重建都具有重要的促进和调节作用。损伤部位的局部生长因子的存在对调节各种神经和非神经细胞的表型改变起着至关重要的作用。神经损伤后，病理环境中的神经营养因子是极度匮乏的，虽然远端神经残端的神经细胞分泌的内源性生长因子能够一定程度上支持轴突再生，但是该种细胞分泌因子的效率及持续时间有限。所以神经损伤修复需要依赖于外源性生长因子的添加。然而外源性生长因子的临床应用还存在许多问题。一方面，大多数生长因子由于半衰期短，扩散快，在体内迅速降解，而另一方面使用高剂量生长因子可能存在未知的安全性问题。与可溶性生长因子不同，模拟生长因子功能的生物活性多肽片段具有理化性质稳定、活性持久、无毒无害、可降解的优点，被广泛用作生长因子的替代物。
基于上述内容，亟待提供一种同时兼具神经仿生定向结构及类神经生长因子的促进神经修复的功能定向纤维复合水凝胶。
发明内容
为了解决上述问题，本申请提出了一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶，所述复合水凝胶由纤维蛋白与多肽复合的定向纤维通过互穿原位交联构成；所述复合水凝胶具有多级取向性结构，复合水凝胶直径为0.5-5.0mm，复合水凝胶由微米纤维丝束构成，微米纤维丝束直径为10μm-100μm；微米纤维丝束由纳米纤维丝束构成，纳米纤维丝束直径为100nm-500nm。
所述复合水凝胶中复合有一种或多种的多肽，所述多肽为自组装多肽；进一步的，所述自组装多肽包括功能多肽；更进一步的，所述功能多肽包括人工合成的层粘连蛋白、纤维粘连蛋白、钙粘着蛋白、神经生长因子、脑源性神经生长因子或血管内皮生长因子的功能活性短肽接枝的自组装多肽。
多肽中功能多肽的含量为10wt％-100wt％。
一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶的制备方法，包括如下步骤：
1)CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温，所述交联溶液中CaCl
2的质量分数为0.5％-3％，凝血酶的含量为4-50Units/mL，将所述交联溶液置于旋转接收盘中待用；
2)将电纺原液进行静电纺丝，电纺原液的添加速度为1.5-3mL/h，加载电压为3-6kV；
3)使用旋转接收盘接收静电纺丝得到的纤维蛋白原纺丝，旋转接收速度为50-100rpm，纤维蛋白原纺丝在交联溶液的作用下得到复合产物；
4)将复合产物继续强化交联，得到所述复合水凝胶。
所述步骤2)中电纺原液的配制方法为：纤维蛋白原溶液与多肽溶液混合均匀，再加入聚氧化乙烯水溶液混合均匀，得到电纺原液。
所述步骤2)中电纺原液的配制方法为：将纤维蛋白原溶液与聚氧化乙烯水溶液混合均匀，得到电纺原液；在步骤3)之后，还包括以下步骤：将复合产物浸润于多肽溶液中，平衡后静置20min。
纤维蛋白原溶液的配制方法为：将纤维蛋白原溶解于纯水中，得到纤维蛋白原溶液，其中纤维蛋白原的质量分数为2％-8％；
聚氧化乙烯水溶液的配制方法为：将聚氧化乙烯溶解于纯水中，得到聚氧化乙烯水溶液，其中，聚氧化乙烯的质量分数为0.1％-0.8％；
多肽溶液的配制方法为：将功能多肽粉末溶解于纯水中，分散后得到多肽溶液；所述多肽溶液的质量分数为0.5％-2％，置于4℃待用。
步骤4)所述交联反应通过多肽自主装交联形成，交联反应包括谷氨酰胺转氨酶催化酰基转移反应的酶交联方法，EDC/NHS催化纤维蛋白与多肽片段形成酰胺键的化学交联方法，采用戊二醛化学交联，采用京尼平化学交联。
所述步骤4)交联反应的条件为：37℃控温交联，磷酸盐缓冲溶液交联，10-30Units/mL谷氨酰胺转氨酶的磷酸盐缓冲溶液交联，浓度为10-60mmol/L的1-乙基-3-(3-二甲基氨丙基)碳二亚胺+浓度为3-30mmol/L的N-羟基琥珀酰亚胺+浓度为50mmol/L的磷酸二氢钾溶液交联其中一种或几种联用。
一种定向纤维蛋白与多肽互穿网络复合水凝胶在神经损伤修复中的应用。
本申请的有益效果在于：
1.本申请通过单通道静电纺丝技术制备的定向纤维蛋白与功能多肽复合水凝胶是一种高含水量的水凝胶材料，由具有高度模拟神经组织的多级取向性纳米纤维束构成，更重要的是同时模拟生长因子功能、具备生物化学活性的功能多肽片段，该定向纤维蛋白与功能多肽复合水凝胶表现出与天然神经组织相类似的成分结构、力学性能、细胞外基质环境及生物活性因子，具有优异的促进神经细胞黏附、神经纤维定向生长，诱导神经和血管再生修复的作用，能应用于脑、脊髓和外周神经损伤的神经再生修复。
2.本申请可以克服现有用于神经损伤修复的导管材料和填充材料生物活性的不足、及目前生长因子使用的安全性差性价比低等问题，提供一种定向纤维蛋白与功能多肽复合水凝胶，可用于神经组织损伤的修复治疗。它是用于神经组织缺损修复的新型复合材料。所述复合水凝胶具有诱导缺损处神经定向生长的微观结构，同时实现功能多肽片段的控制释放，起到类似生长因子的作用，促进神经组织和周围血管再生，同时避免再生的轴突无序生长及外源生长因子添加造成的不可预知及不必要的生理或者病理作用。
3.基于改善神经损伤后促进神经修复的微环境仿生构建策略，本申请构建了具有神经仿生定向结构及类神经生长因子的促进神经修复的功能定向纤维复合水凝胶。所述复合水凝胶 在结构及成分上改善了损伤神经的生长环境，有利于神经细胞的修复及定向轴突的产生。本申请设计结合了定向结构对于神经修复的物理特性及生长因子对神经修复的生物化学特性，制备定向功能纳米纤维水凝胶，弥补现有神经修复材料的结构及功能不足等问题，是一种具有促进神经再生应用前景的复合神经替代物。
4.本申请所要求保护的定向纤维蛋白是通过液态接收的静电纺丝技术制备得到，得到的具有微纳尺度的多级别定向结构，此结构旨在模拟天然的神经纤维定向结构及神经纤维经由神经内膜、神经束膜、神经外膜层级组装的内生结构。不同于以往的非定向的纤维蛋白水凝胶，本申请定向纤维蛋白水凝胶是具有独特的仿生设计结构的仿生水凝胶材料。
5.本申请不同于单纯的自组装多肽水凝胶，将多肽的功能与定向纤维蛋白水凝胶进行互穿网络复合，水凝胶也由原本纳米级别的纤维蛋白变成纳米的复合纤维，因此复合水凝胶材料是由功能的定向纳米纤维逐级定向形成，最终呈现出具有多级定向结构的功能纤维蛋白水凝胶。
6.本申请复合水凝胶材料充分结合了定向纤维蛋白水凝胶与自组装多肽水凝胶的特点，形成具有再生修复特定诱导功能的仿生定向水凝胶材料。不同于单独的上述两种材料，本申请复合水凝胶材料形成了新的纳米功能纤维，水凝胶由新的功能纳米纤维构成。从纤维的构成、结构、形貌特点及功能特性上来说，复合的纳米水凝胶均不同于单独的定向纤维蛋白及自组装功能多肽；在外周神经和脊髓神经修复的实验模型中，复合的定向纤维蛋白水凝胶也明显优于单纯的纤维蛋白和功能多肽的组，具有更加优异的促进神经再生修复的功能作用。
图1为复合水凝胶的实物照片图；
图2为复合水凝胶的扫描电镜图；
图3为复合水凝胶的横截面透射电镜图；
图4为水凝胶接种雪旺细胞3天后的细胞染色结果图；
图5为复合水凝胶移植于动物坐骨神经缺损部位的手术照片；
图6为复合水凝胶移植于动物坐骨神经缺损部位后12周的甲苯胺蓝染色结果图；
图7为复合水凝胶移植于动物脊髓半横断缺损部位的手术照片；
图8为复合水凝胶移植于动物脊髓半横断缺损部位后12周苏木精-伊红染色结果图。
以下结合附图和具体实施例对本申请作进一步的详细说明：
本申请提供了一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶及其制备方法，该定向纤维蛋白与多肽互穿网络复合水凝胶通过静电纺丝技术结合分子自组装、酶交联 或物理及化学交联的方式制备而成。
本申请设计的定向纤维蛋白与功能多肽互穿网络复合水凝胶的设计思路基于天然神经组织的特殊结构及性能。该水凝胶具有多级取向性结构，纤维尺寸从纳米级到微米级。结构由纳米纤维丝束构成，该纳米纤维丝束先构成微米纤维丝束，再由微米纤维丝束构成宏观水凝胶束。纳米纤维丝束的直径为100nm-500nm，微米纤维丝束的直径为10μm-100μm。最后制得的定向纤维蛋白与多肽互穿网络复合水凝胶的直径为0.5-5.0mm。
本申请的定向纤维蛋白与多肽互穿网络复合水凝胶是一种高含水量的水凝胶材料，其组成、结构、力学性能高度仿生天然神经组织的细胞外基质，且具有模拟生长因子功能的多肽片段，具有优异的促进神经纤维定向生长，诱导神经和血管再生的作用。
如图1所示，复合水凝胶呈现直径均匀的定向纤维束外观。
如图2中a和b部分所示，在扫描电镜下观察纤维蛋白纤维排列整齐，呈现多级定向的特点；并且自组装多肽纳米纤维丝束附着在微米纤维丝束的表面，为纤维蛋白的纳米纤维丝束增加多元的表面微结构。
在透射电镜下观察复合水凝胶的横截面，单纯的定向纤维蛋白水凝胶纳米纤维结构紧密，复合水凝胶的纳米纤维呈现更粗的纤维直径和单根纤维内更松散的多孔纳米结构，如图3中a部分所示；并且结合图3中b部分，可以看到自组装多肽纳米纤维穿插于纤维蛋白的纳米纤维内部和表面，形成两者互穿的复合网络结构。
如图4所示，雪旺细胞在复合水凝胶上呈现定向排列的形态，与复合水凝胶自身取向一致，表明复合水凝胶能够诱导雪旺细胞的定向生长。
将本申请制得的复合水凝胶材料应用于大鼠坐骨神经修复，如图5所示，将材料填充在外周神经修复导管内部桥接断端两头的神经，图5中a部分为空白对照的空导管组别，图5中b部分为在空导管内填充本申请复合水凝胶材料进行修复的照片。从图5中b部分照片，可以明显的看出，术后12周后，神经组织已经长到缺损的远端，且成熟的有髓神经密度较高如图6所示，表明本申请材料能够促进外周神经的再生。
将本申请复合水凝胶材料移植于大鼠脊髓T9节段横断缺损部位如图7，12周后脊髓神经组织能够再生于缺损部位，如图8所示为损伤部位组织染色结果。
在本申请的实施方式中，该定向纤维蛋白与功能多肽复合水凝胶的具体直径和长度可以根据手术需要的缺损神经的直径和长度来确定。
本申请用于神经再生的定向纤维蛋白与功能多肽互穿网络复合水凝胶的制备方法包括：
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液，所述电纺原液中纤维蛋白原的质量分数为2％-8％，聚氧化乙烯的质量分数为0.1％-0.8％，将所述电纺原液注入注射器中待用；
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温，所述交联溶液中CaCl
2的质量分数为0.5％-3％，凝血酶的含量为4-50Units/mL，将所述交联溶液置于金属旋转接收盘 中待用；
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原的推注速度为2-3mL/h，加载电压为3-6kV，使用金属旋转接收盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50-100rpm，所述纤维蛋白原纺丝在交联溶液的作用下得到定向纤维蛋白水凝胶，收集得到所需直径的定向纤维蛋白水凝胶；
4)将功能多肽粉末溶解于纯水中，超声分散后得到功能多肽溶液，所述功能多肽溶液的质量分数为0.5％-2％，置于4℃待用；
5)将定向纤维蛋白水凝胶浸润于功能多肽溶液中，平衡后静置20min，通过溶胀及物理吸附使功能多肽与定向纤维蛋白进行互穿，得到物理结合的定向纤维蛋白与功能多肽复合物；
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于交联条件中原位交联，得到交联后的纤维蛋白复合功能多肽的定向纤维水凝胶，上述交联条件包括以下一种或几种：37℃控温交联，磷酸盐缓冲溶液(PBS)溶液交联，10-30Units/mL谷氨酰胺转氨酶的PBS溶液交联和10-60mmol/L1-乙基-3-(3-二甲基氨丙基)碳二亚胺(EDC)+3-30mmol/LN-羟基琥珀酰亚胺(NHS)+50mmol/L磷酸二氢钾溶液交联等。
或
1)将纤维蛋白原溶解于纯水中，纤维蛋白原的质量分数为2％-8％，将聚氧化乙烯溶解于纯水中，质量分数为0.1％-0.8％；将功能多肽粉末溶解于纯水中，超声分散后得到功能多肽溶液，所述功能多肽溶液的质量分数为0.5％-2％，置于4℃待用；
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温，所述交联溶液中CaCl
2的质量分数为0.5％-3％，凝血酶的含量为4-50Units/mL，将所述交联溶液置于金属旋转接收盘中待用；
3)先将纤维蛋白原溶液与功能多肽溶液混合均匀，再加入聚氧化乙烯水溶液混合均匀，得到电纺原液；将注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原的推注速度为1.5-3mL/h，加载电压为3-6kV，使用金属旋转接收盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50-100rpm，复合纤维蛋白纺丝在交联溶液的作用下得到定向复合纤维蛋白水凝胶，收集得到所需直径的定向功能纤维蛋白水凝胶；
4)将上述定向功能纤维蛋白水凝胶在后续交联条件中继续交联孵育，交联条件包括以下一种或几种：37℃控温交联，磷酸盐缓冲溶液(PBS)溶液交联，10-30Units/mL谷氨酰胺转氨酶的PBS溶液交联和10-60mmol/L1-乙基-3-(3-二甲基氨丙基)碳二亚胺(EDC)+3-30mmol/LN-羟基琥珀酰亚胺(NHS)+50mmol/L磷酸二氢钾溶液交联等。
复合纤维水凝胶中复合有一种或多种功能多肽，功能多肽为人工合成的层粘连蛋白(IKVAV，氨基酸序列同缩写)、纤维粘连蛋白(RGD，氨基酸序列同缩写)、钙粘着蛋白(HAVDI，氨基酸序列同缩写)、神经生长因子(CTD,CTDIKGKCTGACDGKQC)、脑源性神经生长因子(RGI,RGIDKRHWNSQ)、血管内皮生长因子(KLT,KLTWQELYQLKYKGI) 的功能活性短肽接枝的自组装多肽(RADA,RADARADARADARADA)、自组装多肽混合物。所述功能多肽片段及可自主装多肽片段不限于此。
下文将结合具体实施例对本申请做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本申请，而不应被解释为对本申请保护范围的限制。凡基于本申请上述内容所实现的技术均涵盖在本申请旨在保护的范围内。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法；下述实施例中所用的试剂、材料等，如无特殊说明，均可从商业途径得到。
实施例1
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液的质量分数均为1％，上述两种自组装多肽含量均为50％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于37℃恒温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶，水凝胶直径为1-2mm，扫描电镜观察复合纤维的微观结构，其纳米纤维直径为100-200nm。
实施例2
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量均为25％，单纯自组装多肽含量为50％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于37℃恒温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶，水凝胶直径为1-2mm，扫描电镜观察复合纤维的微观结构，其纳米纤维直径为100-200nm。
实施例3
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量均为25％，单纯自组装多肽含量为50％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶，水凝胶直径为1-2mm，扫描电镜观察复合纤维的微观结构，其纳米纤维直径为100-200nm。
实施例4
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量均为25％，单纯自组装多肽含量为50％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于10Units/mL谷氨酰胺转氨酶的PBS中室温平衡反应2h，得到稳定的定向纤维蛋白与功能多肽复合水凝胶，水凝胶直径为1-2mm，扫描电镜观察复合纤维的微观结构，其纳米纤维直径为100-200nm。
实施例5
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为4％，聚氧化乙烯的质量分数为0.8％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收 盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量均为50％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡反应30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶，水凝胶直径为1-2mm，扫描电镜观察复合纤维的微观结构，其纳米纤维直径为100-200nm。
实施例6
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量分别为25％和50％，自组装多肽溶液含量为25％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡反应30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例7
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量分别为50％和25％，自组装多肽溶液含量为25％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡反应30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例8
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶 液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的分别接枝了IKVAV和CTD的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液，两种功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述两种功能自组装多肽含量分别为25％和50％，自组装多肽溶液含量为25％，得到的双功能自组装多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述双功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡反应30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例9
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了CTD功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有神经生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为50％，单纯自组装多肽含量为50％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例10
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注 射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了CTD功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有神经生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为30％，单纯自组装多肽含量为70％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例11
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了RGI功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有脑源性神经生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为50％，单纯自组装多肽含量为50％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例12
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了RGI功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有脑源性神经生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为30％，单纯自组装多肽含量为70％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例13
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了KLT功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有血管内皮生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为50％，单纯自组装多肽含量为50％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例14
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了KLT功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有血管内皮生长因子的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为30％，单纯自组装多肽含量为70％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例15
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中 CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了HAVDI功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有钙粘着蛋白功能的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为30％，单纯自组装多肽含量为70％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
(6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例16
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了IKVAV功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有层粘连蛋白功能的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为30％，单纯自组装多肽含量为70％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例17
1)将纤维蛋白原溶解于纯水中，与聚氧化乙烯水溶液混合均匀，得到电纺原液。该电纺原液中纤维蛋白的质量分数为2％，聚氧化乙烯的质量分数为0.5％。随后将该电纺原液注入注射器中待用。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该纤维蛋白原纺丝在交联溶液的作用下得到定向的纤维蛋白水凝胶束。收集直径1mm的定向纤维蛋白水凝胶。
4)将人工合成的接枝了RGD功能多肽的自组装多肽RADA粉末溶解于纯水中，超声分散后得到具有纤维粘连蛋白功能的自组装多肽溶液，功能自组装多肽溶液及自组装多肽溶液的质量分数均为1％，上述功能自组装多肽含量为50％，单纯自组装多肽含量为50％，得到的多肽溶液，置于4℃冰箱待用。
5)将定向纤维蛋白水凝胶浸润于上述功能多肽溶液中，静置20min，得到物理结合的定向纤维蛋白与功能多肽复合水凝胶。
6)将上述物理结合的定向纤维蛋白与功能多肽复合物置于PBS中室温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例18
1)将纤维蛋白原溶解于纯水中，将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)纤维蛋白水溶液与两种功能自组装多肽溶液进行混合，纤维蛋白原浓度为2％，两种多肽溶液的的质量分数均为1％，上述两种自组装多肽含量均为50％；混合溶液与聚氧化乙烯水溶液混合，浓度为0.5％，将上述溶液注入注射器中待用。注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2.5mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该复合纤维蛋白纺丝在交联溶液的作用下得到定向双功能纤维蛋 白水凝胶。收集直径1mm的定向纤维蛋白水凝胶。
4)将上述定向双功能纤维蛋白水凝胶置于37℃恒温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例19
1)将纤维蛋白原溶解于纯水中，将人工合成的分别接枝了RGI和KLT的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后分别得到具有脑源性神经生长因子功能的自组装多肽溶液和具有血管内皮生长因子功能的自组装多肽溶液。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)纤维蛋白水溶液与两种功能自组装多肽溶液进行混合，纤维蛋白原浓度为2％，两种多肽溶液的的质量分数均为1％，上述两种功能自组装多肽含量分别为25％和50％，自组装多肽溶液含量为25％；混合溶液与聚氧化乙烯水溶液混合，浓度为0.5％，将上述溶液注入注射器中待用。注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2.5mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该复合纤维蛋白纺丝在交联溶液的作用下得到定向双功能纤维蛋白水凝胶。收集直径1mm的定向纤维蛋白水凝胶。
4)将上述定向双功能纤维蛋白水凝胶置于37℃恒温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
实施例20
1)将纤维蛋白原溶解于纯水中，将人工合成的接枝RGD的功能多肽的自组装多肽RADA粉末分别溶解于纯水中，超声分散后得到具有纤维粘连蛋白功能的自组装多肽溶液。
2)将CaCl
2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温待用。该交联溶液中CaCl
2的质量分数为0.5％，凝血酶的含量为4Units/mL。随后将该交联溶液置于金属旋转接收盘中待用。
3)纤维蛋白水溶液与功能自组装多肽溶液进行混合，纤维蛋白原浓度为2％，多肽溶液的的质量分数均为1％，上述功能自组装多肽含量50％，自组装多肽溶液含量为50％；混合溶液与聚氧化乙烯水溶液混合，浓度为0.5％，将上述溶液注入注射器中待用。注入注射器的电纺原液在推进泵的作用下进行静电纺丝，调整纤维蛋白原溶液的推注速度为2.5mL/h，加载电压为4kV，使其得到连续的稳定的纤维蛋白原纺丝。使用金属选转盘接收静电纺丝得到的纤维蛋白原纺丝，调整旋转接收速度为50rpm，该复合纤维蛋白纺丝在交联溶液的作用下得到定向功能纤维蛋白水凝胶。收集直径1mm的定向纤维蛋白水凝胶。
4)将上述定向功能纤维蛋白水凝胶置于37℃恒温平衡30min，得到稳定的定向纤维蛋白与功能多肽复合水凝胶。
以上，对本申请的实施方式进行了说明。但是，本申请不限定于上述实施方式。凡在本申请的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
Claims (13)
- 一种用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶，其中，所述复合水凝胶由纤维蛋白与多肽复合的定向纤维通过互穿原位交联构成；所述复合水凝胶具有多级取向性结构，复合水凝胶直径为0.5-5.0mm，复合水凝胶由微米纤维丝束构成，微米纤维丝束直径为10μm-100μm；微米纤维丝束由纳米纤维丝束构成，纳米纤维丝束直径为100nm-500nm。
- 根据权利要求1所述的复合水凝胶，其中，所述复合水凝胶中复合有一种或多种的多肽，所述多肽为自组装多肽。
- 根据权利要求2所述的复合水凝胶，其中，所述自组装多肽包括功能多肽。
- 根据权利要求3所述的复合水凝胶，其中，所述功能多肽包括人工合成的层粘连蛋白、纤维粘连蛋白、钙粘着蛋白、神经生长因子、脑源性神经生长因子或血管内皮生长因子的功能活性短肽接枝的自组装多肽。
- 根据权利要求2-4任一项所述的复合水凝胶，其中，多肽中功能多肽的含量为10wt％-100wt％。
- 一种权利要求1-5中任一项所述的用于神经再生的定向纤维蛋白与多肽互穿网络复合水凝胶的制备方法，其中，包括如下步骤：1)CaCl 2水溶液与凝血酶溶液混合得到交联溶液并在37℃保温，所述交联溶液中CaCl 2的质量分数为0.5％-3％，凝血酶的含量为4-50Units/mL，将所述交联溶液置于旋转接收盘中待用；2)将电纺原液进行静电纺丝，电纺原液的添加速度为1.5-3mL/h，加载电压为3-6kV；3)使用旋转接收盘接收静电纺丝得到的纤维蛋白原纺丝，旋转接收速度为50-100rpm，纤维蛋白原纺丝在交联溶液的作用下得到复合产物；4)将复合产物继续强化交联，得到所述复合水凝胶。
- 根据权利要求6所述的复合水凝胶的制备方法，其中，所述步骤2)中电纺原液的配制方法为：纤维蛋白原溶液与多肽溶液混合均匀，再加入聚氧化乙烯水溶液混合均匀，得到电纺原液。
- 根据权利要求6所述的复合水凝胶的制备方法，其中，所述步骤2)中电纺原液的配制方法为：将纤维蛋白原溶液与聚氧化乙烯水溶液混合均匀，得到电纺原液；在步骤3)之后，还包括以下步骤：将复合产物浸润于多肽溶液中，平衡后静置20min。
- 根据权利要求7或8所述的复合水凝胶的制备方法，其中，纤维蛋白原溶液的配制方法为：将纤维蛋白原溶解于纯水中，得到纤维蛋白原溶液，其中纤维蛋白原的质量分数为2％-8％。
- 根据权利要求7或8所述的复合水凝胶的制备方法，其中，聚氧化乙烯水溶液的配制方法为：将聚氧化乙烯溶解于纯水中，得到聚氧化乙烯水溶液，其中，聚氧化乙烯的质量分数为0.1％-0.8％。
- 根据权利要求7或8所述的复合水凝胶的制备方法，其中，多肽溶液的配制方法为： 将多肽粉末溶解于纯水中，分散后得到多肽溶液；所述多肽溶液的质量分数为0.5％-2％，置于4℃待用。
- 根据权利要求6所述的复合水凝胶的制备方法，其中，所述步骤4)中的强化交联可选自37℃控温交联、磷酸盐缓冲溶液交联、10-30Units/mL谷氨酰胺转氨酶的磷酸盐缓冲溶液交联、浓度为10-60mmol/L的1-乙基-3-(3-二甲基氨丙基)碳二亚胺+浓度为3-30mmol/L的N-羟基琥珀酰亚胺+浓度为50mmol/L的磷酸二氢钾溶液交联中一种或几种联用。
- 一种权利要求1-5任一项所述定向纤维蛋白与多肽互穿网络复合水凝胶在神经损伤修复中的应用。
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