复合丝素蛋白神经导管的制备及周围神经缺损的修复研究
发布时间：2018-07-31 08:47
【摘要】：周围神经损伤是临床上常见的病症,其修复与再生是神经科学领域的研究热点。当损伤距离较短时,周围神经能自我修复,但是长距离缺损,必须借助神经移植物才能完成修复。作为“金标准”的自体神经移植存在来源匮乏、供体与损伤神经尺寸不匹配等不足,因此寻求合适的人工神经移植物,引导、促进神经再生,加快功能重建是科研人员努力的目标。理想的人工神经移植物必须具备良好的生物相容性和与植入组织匹配的力学性能。大量研究表明蚕丝的丝素蛋白具有良好的生物相容性,但蚕丝在脱胶过程中力学性能大幅度下降。为构建符合要求的人工神经移植物,本课题以天然蚕丝为基本原料,将传统的编织法与静电纺丝法相结合,制备兼具良好生物相容性和力学性能的具有复合结构的丝素蛋白神经导管(CSF-NGCs),对导管进行生物安全性评价,并将其用于修复大鼠10 mm坐骨神经缺损,通过一系列方法评价其修复效果。主要研究内容和结论如下:利用静电纺丝法制备CSF-NGCs的内层和外层,借助扫描电子显微镜(SEM)研究了溶液浓度、电压、推进速度对静电纺纳米纤维形貌和尺寸的影响,筛选出最优工艺参数为:溶液浓度:18%,电压:21 kV,推进速度:0.2 mL/h。用自改装的编织机编织丝素纤维网,通过改变携纱器的运转速度和编织物向上提拉速度,获得具有不同编织角和编织密度的丝素网。测试所制备导管的拉伸性能、抗手术缝合线强度以及抗压性能,分析了编织角、编织密度对三种力学性能的影响。通过比较最终用于后续实验的CSF-NGCs拉伸时最大荷重为16.3 N,将导管拉破时拉力与2倍管壁厚度的比值Fpull-out/2t值是3.5N/mm,抗压试验中形变50%时对应的荷重达2.1 N,各项指标都与文献报道结果相当,同时明显优于单纯静电纺丝素导管。后续缝合术和体内试验证实了这一点。根据实验操作要求与力学性能测试结果,确定编织最佳工艺参数为:携纱器运行速度:3.6 rpm,编织物上提速度:4.5 cm/min。测定CSF-NGCs的壁厚、表面形貌、孔隙率、吸水性、通透性,结果表明CSF-NGCs具有三维多孔纳米结构,吸水性、渗透性良好。对CSF-NGCs进行体外模拟降解,分析了降解过程中丝素力学性能、蛋白结构、质量等的变化,结果表明CSF-NGCs在体外蛋白酶XIV溶液中可降解。通过静电纺丝法制备了静电纺丝素纳米纤维膜,将膜与雪旺氏细胞及背根神经节共培养,培养3天和5天时拍照,并进行扫描电镜和免疫荧光染色观察,MTT法检测细胞活力,同时用实时PCR检测雪旺氏细胞BDNF和NGF mRNA的表达及释放情况。结果表明静电纺丝素纳米纤维膜与周围神经组织、细胞具有良好的生物相容性,为神经导管的构建及体内研究奠定了基础。按照GB/T 16886规定的方法,通过遗传实验(包括Ames试验、小鼠骨髓嗜多染红细胞(PCE)微核试验、小鼠精子畸变实验)、体外细胞毒性实验、皮内实验、迟发型超敏反应试验、皮下植入局部反应试验、急性全身毒性实验对所制备的CSF-NGCs进行了生物安全性评价,结果表明CSF-NGCs无遗传毒性和细胞毒性、对皮肤无刺激性和潜在致敏性、无急性全身毒性作用,生物相容性好,可生物降解,符合GB/T 16886关于医疗器械生物学评价的相关标准。将制备的CSF-NGCs用于桥接SD大鼠坐骨神经10 mm缺损(导管组),另设自体组和缺损组。术后1、3、6个月,以Catwalk步态仪检测、分析其运动功能的恢复情况;术后3、6个月行热痛觉测定、运用光镜观察靶肌形态并利用图像分析系统进行计量分析;术后6个月进行电生理检测,对再生神经进行免疫组化染色,运用光镜和电镜技术观察再生神经形貌并进行统计分析。结果:移植术后,随着时间推移,导管组和自体组各方面功能都在逐步恢复,自体组恢复较快,导管组恢复较慢,3个月时两组间存在较大差异,但术后6个月,所有检测项目结果与自体组的差异都不具统计学意义,而与缺损组间差异显著。术后6个月具体数据:(1)自体组、导管组、缺损组步态规律指数RI分别为:85%、79%、29%(95%以上为正常),坐骨神经功能指数SFI分别为-55、-58、-85(0为完全恢复,-100为完全损伤);(2)用爪痛测试仪记录大鼠热痛阈潜伏期,非术侧、自体组、导管组平均潜伏期分别为7.78 s、9.15 s、10.23 s,缺损组大鼠脚基本不动,所以时间都超过设定值20.1 s;(3)电生理检测:自体组、导管组的腓肠肌复合肌动作电位(CMAP)波幅分别相当于非术侧的72.7%和65.3%,导管组、自体组和非术侧的神经传导速度分别为28.10±4.03 m/s、35.57±3.49 m/s和50.00±3.18 m/s,缺损组未记录到CMAP;(4)自体组、导管组、缺损组腓肠肌湿重比分别为0.64、0.57、0.18,肌纤维平均横截面积分别相当于非术侧的82.9%、74.8%、9.8%;(5)免疫组化结果显示导管组和自体组的轴突排布都比较乱,直径也较非术侧小,缺损组只有极少量神经纤维;(6)电镜观察结果:导管组和自体组的有髓神经纤维都比较紧密,虽然髓鞘厚度不及非术侧,但有完整的基底膜。非术侧、自体组、导管组髓鞘厚度分别为:1.38?m、0.78?m、0.65?m,神经纤维直径分别为2.31?m、1.62?m、1.51?m,缺损组几乎无有髓神经。综上所述,各实验结果表明术后6个月,导管组大鼠运动功能、感觉功能都恢复良好,修复效果和自体组相当。本课题的研究工作为制备人工神经导管提供了新思路,新方法,相关结果为新制备导管在组织工程领域的进一步研究与应用提供了理论基础和依据。
[Abstract]:Peripheral nerve injury is a common clinical disease, and its repair and regeneration is a hot spot in the field of neuroscience. When the injury distance is short, the peripheral nerve can repair itself, but the long distance defect must be repaired with the aid of the nerve graft. There is a shortage of sources, donor and injury as the "gold standard" of autologous nerve transplantation. Therefore, it is the goal of researchers to seek suitable artificial nerve graft, guide, promote nerve regeneration and accelerate functional reconstruction. Ideal artificial nerve graft must have good biocompatibility and the ability to match the implanted tissue. A large number of studies show silk fibroin albumen. It has good biocompatibility, but the mechanical properties of silk in the process of degumming are greatly reduced. In order to construct the artificial neural grafts that meet the requirements, this subject uses natural silk as the basic raw material to combine the traditional knitting method with the electrostatic spinning method to prepare a composite silk egg with a composite structure with good biocompatibility and mechanical properties. The white nerve conduit (CSF-NGCs) was used to evaluate the biological safety of the catheter and to repair the 10 mm sciatic nerve defect of the rat and evaluate the repair effect by a series of methods. The main contents and conclusions are as follows: the inner and outer layers of CSF-NGCs were prepared by the electrostatic spinning method, and the concentration of the solution was studied by scanning electron microscope (SEM). The effect of voltage and propulsion speed on the morphology and size of electrospun nanofibers was selected. The optimum process parameters were as follows: solution concentration: 18%, voltage: 21 kV, propulsion speed: 0.2 mL/h. weave silk fibroin with self modified braiding machine, and obtain different knitting angles and weaving angles by changing the speed of the yarn carrying device and the speed of the knitting fabric to the up drawing speed. The silk net of the woven density. Test the tensile properties, the strength of the surgical suture and the compressive strength. The influence of the braiding angle and the braiding density on the three mechanical properties was analyzed. The maximum weight of the three kinds of mechanical properties was 16.3 N when the final CSF-NGCs was used for the subsequent experiment. The ratio of the tensile force to the 2 times the thickness of the tube wall when the catheter was pulled out was Fpull. The -out/2t value is 3.5N/mm, and the corresponding load of the compression test is 2.1 N when the deformation is 50%. Each index is equivalent to the literature report, and it is obviously superior to the pure electrospun silk conduit. The following suture and in vivo test confirm this point. According to the experimental operation requirements and the mechanical performance test results, the best weaving process parameters are determined to be carried out. The running speed of the yarn is 3.6 rpm, the speed of lifting on the fabric: 4.5 cm/min. to determine the wall thickness, surface morphology, porosity, water absorption and permeability of CSF-NGCs. The results show that CSF-NGCs has three-dimensional porous nanostructure, water absorbability and permeability. The mechanical properties, protein structure and quality of the fibroin in the degradation process are analyzed in vitro. The results showed that the CSF-NGCs was degradable in the protease XIV solution in vitro. Electrospun silk fibroin nanofibrous membrane was prepared by electrostatic spinning method. The membrane was co cultured with Schwann cell and dorsal root ganglion and cultured for 3 days and 5 days. The scanning electron microscope and immunofluorescence staining were used to detect the activity of the cell, and the cell viability was detected by MTT method. The expression and release of BDNF and NGF mRNA in Schwann cells were detected by real time PCR. The results showed that the electrospun fibroin nanofiber membrane had good biocompatibility with the peripheral nerve tissue and cells. It laid the foundation for the construction of nerve conduit and in vivo research. According to the method of GB/T 16886, the genetic experiment (including Ames test, small) Mouse bone marrow polychromatic erythrocyte (PCE) micronucleus test, mouse sperm aberration test, in vitro cytotoxicity test, intradermal experiment, delayed type hypersensitivity test, subcutaneous implantation local reaction test and acute systemic toxicity test were used to evaluate the biological safety of the prepared CSF-NGCs. The results showed that CSF-NGCs had no hereditary toxicity and cytotoxicity. No irritation and potential sensitivities to the skin, no acute systemic toxicity, good biocompatibility and biodegradability, which accords with the related standards of GB/T 16886 on biological evaluation of medical instruments. The prepared CSF-NGCs was used to bridge the 10 mm defect of the sciatic nerve of SD rats (catheter group), and also set up the autologous group and the defect group. After 1,3,6 months, Catwalk A gait instrument was used to detect the recovery of the motor function. After 3,6 months, the heat pain was measured, the shape of the target muscle was observed by light microscope and the image analysis system was used for measurement and analysis. The electrophysiological examination was carried out for 6 months after the operation, the regenerated nerve was immunohistochemical staining, and the morphologies of the regenerated nerve were observed by light microscopy and electron microscopy. Statistical analysis. Results: after the transplantation, the functions of the catheter group and the autologous group were gradually restored with time. The autologous group recovered quickly and the catheter group recovered slowly. There was a big difference between the two groups at 3 months, but 6 months after the operation, the difference between the results of all the test items and the autologous group was not statistically significant, but the difference from the defect group was not significant. 6 months after operation specific data: (1) the gait regularity index RI of autologous group, catheter group and defect group were 85%, 79%, 29% (95% above normal), the sciatic nerve function index SFI was -55, -58, -85 (0 was completely recovered, -100 was completely damaged); (2) the latent period of rat thermal pain threshold was recorded by claw pain tester, non operative, autologous group and catheter group were recorded. The average latency was 7.78 s, 9.15 s, 10.23 s, and the foot of the defect group was basically not moving, so the time exceeded the set value of 20.1 s; (3) electrophysiological test: the autologous group, the gastrocnemius muscle action potential (CMAP) amplitude of the catheter group was equivalent to the non operative 72.7% and 65.3%, the catheter group, the autologous group and the non lateral nerve conduction velocity were respectively 28.10 + 4.03 m/s, 35.57 + 3.49 m/s and 50 + 3.18 m/s, the defect group did not record CMAP; (4) the wet weight ratio of the gastrocnemius muscle in the autologous group, the catheter group and the defect group was 0.64,0.57,0.18, the average cross section of the muscle fiber was equivalent to 82.9%, 74.8%, 9.8% of the non operative side, and the results of the exempting group showed that the axon arrangement in the catheter group and the autologous group were all in disorder. There were only a few nerve fibers in the defect group. (6) the results of electron microscope observation: the medullary nerve fibers in the catheter group and the autologous group were close, although the thickness of the myelin sheath was less than the non operative side, but there was a complete basement membrane. The non operative, autologous and catheter groups were 1.38? M, 0.78? M, 0.65? M, and the diameter of nerve fibers, respectively. For 2.31? M, 1.62? M, 1.51? M, there was almost no myelinated nerve in the defect group. In summary, the experimental results showed that the motor function and sensory function of the rats in the catheter group recovered well and the effect was equivalent to the autologous group at 6 months after the operation. The research work of this subject provided new ideas, new methods, and related results for the preparation of artificial nerve conduits. The tube provides a theoretical basis and basis for further research and application in the field of tissue engineering.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R745
本文编号：2155057
[Abstract]:Peripheral nerve injury is a common clinical disease, and its repair and regeneration is a hot spot in the field of neuroscience. When the injury distance is short, the peripheral nerve can repair itself, but the long distance defect must be repaired with the aid of the nerve graft. There is a shortage of sources, donor and injury as the "gold standard" of autologous nerve transplantation. Therefore, it is the goal of researchers to seek suitable artificial nerve graft, guide, promote nerve regeneration and accelerate functional reconstruction. Ideal artificial nerve graft must have good biocompatibility and the ability to match the implanted tissue. A large number of studies show silk fibroin albumen. It has good biocompatibility, but the mechanical properties of silk in the process of degumming are greatly reduced. In order to construct the artificial neural grafts that meet the requirements, this subject uses natural silk as the basic raw material to combine the traditional knitting method with the electrostatic spinning method to prepare a composite silk egg with a composite structure with good biocompatibility and mechanical properties. The white nerve conduit (CSF-NGCs) was used to evaluate the biological safety of the catheter and to repair the 10 mm sciatic nerve defect of the rat and evaluate the repair effect by a series of methods. The main contents and conclusions are as follows: the inner and outer layers of CSF-NGCs were prepared by the electrostatic spinning method, and the concentration of the solution was studied by scanning electron microscope (SEM). The effect of voltage and propulsion speed on the morphology and size of electrospun nanofibers was selected. The optimum process parameters were as follows: solution concentration: 18%, voltage: 21 kV, propulsion speed: 0.2 mL/h. weave silk fibroin with self modified braiding machine, and obtain different knitting angles and weaving angles by changing the speed of the yarn carrying device and the speed of the knitting fabric to the up drawing speed. The silk net of the woven density. Test the tensile properties, the strength of the surgical suture and the compressive strength. The influence of the braiding angle and the braiding density on the three mechanical properties was analyzed. The maximum weight of the three kinds of mechanical properties was 16.3 N when the final CSF-NGCs was used for the subsequent experiment. The ratio of the tensile force to the 2 times the thickness of the tube wall when the catheter was pulled out was Fpull. The -out/2t value is 3.5N/mm, and the corresponding load of the compression test is 2.1 N when the deformation is 50%. Each index is equivalent to the literature report, and it is obviously superior to the pure electrospun silk conduit. The following suture and in vivo test confirm this point. According to the experimental operation requirements and the mechanical performance test results, the best weaving process parameters are determined to be carried out. The running speed of the yarn is 3.6 rpm, the speed of lifting on the fabric: 4.5 cm/min. to determine the wall thickness, surface morphology, porosity, water absorption and permeability of CSF-NGCs. The results show that CSF-NGCs has three-dimensional porous nanostructure, water absorbability and permeability. The mechanical properties, protein structure and quality of the fibroin in the degradation process are analyzed in vitro. The results showed that the CSF-NGCs was degradable in the protease XIV solution in vitro. Electrospun silk fibroin nanofibrous membrane was prepared by electrostatic spinning method. The membrane was co cultured with Schwann cell and dorsal root ganglion and cultured for 3 days and 5 days. The scanning electron microscope and immunofluorescence staining were used to detect the activity of the cell, and the cell viability was detected by MTT method. The expression and release of BDNF and NGF mRNA in Schwann cells were detected by real time PCR. The results showed that the electrospun fibroin nanofiber membrane had good biocompatibility with the peripheral nerve tissue and cells. It laid the foundation for the construction of nerve conduit and in vivo research. According to the method of GB/T 16886, the genetic experiment (including Ames test, small) Mouse bone marrow polychromatic erythrocyte (PCE) micronucleus test, mouse sperm aberration test, in vitro cytotoxicity test, intradermal experiment, delayed type hypersensitivity test, subcutaneous implantation local reaction test and acute systemic toxicity test were used to evaluate the biological safety of the prepared CSF-NGCs. The results showed that CSF-NGCs had no hereditary toxicity and cytotoxicity. No irritation and potential sensitivities to the skin, no acute systemic toxicity, good biocompatibility and biodegradability, which accords with the related standards of GB/T 16886 on biological evaluation of medical instruments. The prepared CSF-NGCs was used to bridge the 10 mm defect of the sciatic nerve of SD rats (catheter group), and also set up the autologous group and the defect group. After 1,3,6 months, Catwalk A gait instrument was used to detect the recovery of the motor function. After 3,6 months, the heat pain was measured, the shape of the target muscle was observed by light microscope and the image analysis system was used for measurement and analysis. The electrophysiological examination was carried out for 6 months after the operation, the regenerated nerve was immunohistochemical staining, and the morphologies of the regenerated nerve were observed by light microscopy and electron microscopy. Statistical analysis. Results: after the transplantation, the functions of the catheter group and the autologous group were gradually restored with time. The autologous group recovered quickly and the catheter group recovered slowly. There was a big difference between the two groups at 3 months, but 6 months after the operation, the difference between the results of all the test items and the autologous group was not statistically significant, but the difference from the defect group was not significant. 6 months after operation specific data: (1) the gait regularity index RI of autologous group, catheter group and defect group were 85%, 79%, 29% (95% above normal), the sciatic nerve function index SFI was -55, -58, -85 (0 was completely recovered, -100 was completely damaged); (2) the latent period of rat thermal pain threshold was recorded by claw pain tester, non operative, autologous group and catheter group were recorded. The average latency was 7.78 s, 9.15 s, 10.23 s, and the foot of the defect group was basically not moving, so the time exceeded the set value of 20.1 s; (3) electrophysiological test: the autologous group, the gastrocnemius muscle action potential (CMAP) amplitude of the catheter group was equivalent to the non operative 72.7% and 65.3%, the catheter group, the autologous group and the non lateral nerve conduction velocity were respectively 28.10 + 4.03 m/s, 35.57 + 3.49 m/s and 50 + 3.18 m/s, the defect group did not record CMAP; (4) the wet weight ratio of the gastrocnemius muscle in the autologous group, the catheter group and the defect group was 0.64,0.57,0.18, the average cross section of the muscle fiber was equivalent to 82.9%, 74.8%, 9.8% of the non operative side, and the results of the exempting group showed that the axon arrangement in the catheter group and the autologous group were all in disorder. There were only a few nerve fibers in the defect group. (6) the results of electron microscope observation: the medullary nerve fibers in the catheter group and the autologous group were close, although the thickness of the myelin sheath was less than the non operative side, but there was a complete basement membrane. The non operative, autologous and catheter groups were 1.38? M, 0.78? M, 0.65? M, and the diameter of nerve fibers, respectively. For 2.31? M, 1.62? M, 1.51? M, there was almost no myelinated nerve in the defect group. In summary, the experimental results showed that the motor function and sensory function of the rats in the catheter group recovered well and the effect was equivalent to the autologous group at 6 months after the operation. The research work of this subject provided new ideas, new methods, and related results for the preparation of artificial nerve conduits. The tube provides a theoretical basis and basis for further research and application in the field of tissue engineering.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R745
【参考文献】
相关期刊论文 前1条
1 姚玉娜,刘萍,王淑娥,朱蕾,董怀军;苦参素对小鼠的急性毒性和外周血红细胞微核和精子畸变试验[J];癌变.畸变.突变;2004年02期
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