聚苯胺和玉米醇溶蛋白管性能、制备以及在神经损伤修复的研究
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摘要
本文主要分为三个部分:
第一部分是种子细胞的分离培养。此部分通过对神经进行预变性处理,从成年SD大鼠的坐骨神经内分离扩增出雪旺细胞,为后续工作提供种子细胞。
第二部分对聚苯胺作为植入式电极涂层材料进行评价。实验表明:1、聚苯胺涂层以纳米球形颗粒(20nm～40nm)形态界面聚合于铂电极表面;2、在体外长期电刺激模型中,此涂层保护电极抵抗电流腐蚀效果显著,并长期稳定。涂层整体完整,无脱落、开裂,纳米球形颗粒微观形貌亦保持,显示出良好的抗腐蚀性和稳定性;3、在SD大鼠视网膜匀浆电刺激模型中,聚苯胺涂层有效抑制电极对网膜匀浆组织的非特异性吸附,并保护其所在组织遭受电刺激诱发的脂质过氧化的攻击,展现出良好的组织保护作用。
第三部分对玉米醇溶蛋白管的制备及其对SD大鼠5mm坐骨神经缺损的修复作用进行了初步研究。
第一节重点探讨玉米醇溶蛋白神经导管的制备及基本性能评价,就其导电性、通透性、生物活性、力学性能等展开研究。自主研发导管制备方法——浸提法,并申请中国专利保护。结果表明: 1、玉米醇溶蛋白及其降解产物具有良好的细胞相容性,PC12细胞对其降解产物存在剂量依赖性;2、导管管壁具有选择通透性;管内壁涂覆具有缓释潜能的纳米级玉米醇溶蛋白微球;管体力学性能适配手术操作和体内植入;3、导管导电性通过涂覆聚苯胺来实现。聚苯胺本身具有良好的细胞相容性,但其降解产物在一定浓度下对细胞的增殖有抑制作用;4、导管生物活性可通过接种细胞或细胞外基质来实现。
第二节建立SD大鼠5mm坐骨神经缺损动物模型,评价无生物活性导管和导电导管的实际修复功效。结果显示,无生物活性导管和导电导管均能在4周内成功桥接SD大鼠5mm坐骨神经缺损,再生轴突长过神经缺损与远端神经相连。这为更好的修复周围神经提供了可能。
本文的创新点主要有:
一、将聚苯胺用作植入式电极涂层,首次在SD大鼠视网膜匀浆体系下评价聚苯胺涂层的保护作用。
1、聚苯胺涂层具有纳米级微观形貌,球形纳米颗粒形态展现出良好的稳定性;
2、在体外电刺激模型中,聚苯胺涂层保护电极抗电流腐蚀效果显著,不仅涂层长期稳定,而且纳米级微观形貌保持良好;
3、在SD大鼠视网膜匀浆电刺激模型中,聚苯胺涂层有效减少电极对网膜组织碎片的非特异性吸附,从而降低炎症反应程度;此外还能明显保护所在组织遭受电刺激引发的脂质过氧化攻击。
二、首次将玉米醇溶蛋白引入周围神经损伤领域,并将其制备成神经导管用于修复神经损伤。玉米醇溶蛋白管及其制备方法申请中国专利保护。
1、玉米醇溶蛋白神经导管具有良好的细胞相容性。导管植入体内后可被降解吸收;体外细胞实验显示PC12细胞对玉米醇溶蛋白管的降解产物存在剂量依赖性。
2、自主研发导管的制备方法。此法操作简单,可重复性强,适用范围广。可制备不同管径(>0.28mm)、管长和壁厚(>18μm)的组织工程用导管。
3、首次将玉米醇溶蛋白与聚苯胺相结合,实现导电导管的制备。为损伤神经早期功能恢复提供可行性。
The main contents of the thesis are as follows:
PART 1: In vitro expansion of Schwann cells from the predegeneratedsciatic nerves of adult SD rat. This part was a prepation of seeding cells forlater investigaion.
PART 2: Evaluation of polyaniline (PANi) as implanted electrodecoatings. It revealed that 1) PANi was in situ polymerized on platinum (Pt)electrodes with nanoparticles from 20nm to 40nm; 2) in long-term electricalstimulation model, PANi coatings showed excellent anticorrosion propertyduring 6 months electrical stimulation, as well as the intactness and thestability; 3) in SD rat retinal homogenate stimulation model, PANi coatingsdramatically protected electrodes and surrounding tissue against nonspecificadsorption and peroxidation, respectively.
PART 3: The fabrication and the investigation of zein conduits in SD rat5mm sciatic nerve regeneration.
The first part was the fabrication and characterization of zein conduits,focus on the conductivity, permeability, bioactivity, mechanical property, etc.The zein conduits and its fabrication were applied China Patent. It indicatedthat 1) zein and its degraded products are biocompatible on PC12 cells; 2) zein conduits were selectively permeable; inner surface coated withnanoscaled microspheres, which was releasable; mechanical property wassuitable for operation and implant; 3) the conductivity of zein conduits wasobtained from PANi coating. PANi are biocompatible on PC12 cells, ratherthan its degraded products, which hamper the cell proliferation in specificcontent; 4) the bioactivity was obtained from implant cells or ECM.The second part was the evaluation of non-bioactive conductive zeinconduits in SD rat model with 5mm sciatic nerve gaps. It showed thatregenerated axons accomplished to connect the distal nerve ends in 4 weeksin both zein conduits and conductive zein conduits. It was feasible inperipheral nerve restoration.
The innovations were mainly in two aspects:
1. Utilization of PANi as coating materials to the implanted electrodes. Itis the first time to evaluate the PANi coating properties in SD rat retinalhomogenate model.
1) The morphology of PANi coating was nanoparticles ranging from20nm to 40nm, which exhibited excellent properties on stability;
2) In long-term electrical stimulation system, the PANi coating showedsplendid anticorrosion property in long-term electrical stimulation, as well asthe intactness and the stability;
3) In the SD rat retinal homogenate electrical stimulation model, thePANi coatings dramatically protected electrodes against nonspecificadsorption, which reduced the inflammation, as well as the protection ofsurrounding tissue from peroxidation.
2. It is firstly to introduce zein into peripheral nerve restoration tofabricate nerve conduits. The zein conduits and its fabrication method were applied China Patent.
1) Zein conduits were biocompatible and biodegradable. PC12 cells relyon the zein degraded products.
2) The procedure of fabrication of zein conduits was simple and wideuse, which could obtain conduits with controllable inner dimmers (>0.28μm),length and depth (>18μm) for tissue engineering.
3) Combined zein with PANi to obtain conductive zein conduits, firsly,for nerve repair and restoration.
第一部分是种子细胞的分离培养。此部分通过对神经进行预变性处理,从成年SD大鼠的坐骨神经内分离扩增出雪旺细胞,为后续工作提供种子细胞。
第二部分对聚苯胺作为植入式电极涂层材料进行评价。实验表明:1、聚苯胺涂层以纳米球形颗粒(20nm～40nm)形态界面聚合于铂电极表面;2、在体外长期电刺激模型中,此涂层保护电极抵抗电流腐蚀效果显著,并长期稳定。涂层整体完整,无脱落、开裂,纳米球形颗粒微观形貌亦保持,显示出良好的抗腐蚀性和稳定性;3、在SD大鼠视网膜匀浆电刺激模型中,聚苯胺涂层有效抑制电极对网膜匀浆组织的非特异性吸附,并保护其所在组织遭受电刺激诱发的脂质过氧化的攻击,展现出良好的组织保护作用。
第三部分对玉米醇溶蛋白管的制备及其对SD大鼠5mm坐骨神经缺损的修复作用进行了初步研究。
第一节重点探讨玉米醇溶蛋白神经导管的制备及基本性能评价,就其导电性、通透性、生物活性、力学性能等展开研究。自主研发导管制备方法——浸提法,并申请中国专利保护。结果表明: 1、玉米醇溶蛋白及其降解产物具有良好的细胞相容性,PC12细胞对其降解产物存在剂量依赖性;2、导管管壁具有选择通透性;管内壁涂覆具有缓释潜能的纳米级玉米醇溶蛋白微球;管体力学性能适配手术操作和体内植入;3、导管导电性通过涂覆聚苯胺来实现。聚苯胺本身具有良好的细胞相容性,但其降解产物在一定浓度下对细胞的增殖有抑制作用;4、导管生物活性可通过接种细胞或细胞外基质来实现。
第二节建立SD大鼠5mm坐骨神经缺损动物模型,评价无生物活性导管和导电导管的实际修复功效。结果显示,无生物活性导管和导电导管均能在4周内成功桥接SD大鼠5mm坐骨神经缺损,再生轴突长过神经缺损与远端神经相连。这为更好的修复周围神经提供了可能。
本文的创新点主要有:
一、将聚苯胺用作植入式电极涂层,首次在SD大鼠视网膜匀浆体系下评价聚苯胺涂层的保护作用。
1、聚苯胺涂层具有纳米级微观形貌,球形纳米颗粒形态展现出良好的稳定性;
2、在体外电刺激模型中,聚苯胺涂层保护电极抗电流腐蚀效果显著,不仅涂层长期稳定,而且纳米级微观形貌保持良好;
3、在SD大鼠视网膜匀浆电刺激模型中,聚苯胺涂层有效减少电极对网膜组织碎片的非特异性吸附,从而降低炎症反应程度;此外还能明显保护所在组织遭受电刺激引发的脂质过氧化攻击。
二、首次将玉米醇溶蛋白引入周围神经损伤领域,并将其制备成神经导管用于修复神经损伤。玉米醇溶蛋白管及其制备方法申请中国专利保护。
1、玉米醇溶蛋白神经导管具有良好的细胞相容性。导管植入体内后可被降解吸收;体外细胞实验显示PC12细胞对玉米醇溶蛋白管的降解产物存在剂量依赖性。
2、自主研发导管的制备方法。此法操作简单,可重复性强,适用范围广。可制备不同管径(>0.28mm)、管长和壁厚(>18μm)的组织工程用导管。
3、首次将玉米醇溶蛋白与聚苯胺相结合,实现导电导管的制备。为损伤神经早期功能恢复提供可行性。
The main contents of the thesis are as follows:
PART 1: In vitro expansion of Schwann cells from the predegeneratedsciatic nerves of adult SD rat. This part was a prepation of seeding cells forlater investigaion.
PART 2: Evaluation of polyaniline (PANi) as implanted electrodecoatings. It revealed that 1) PANi was in situ polymerized on platinum (Pt)electrodes with nanoparticles from 20nm to 40nm; 2) in long-term electricalstimulation model, PANi coatings showed excellent anticorrosion propertyduring 6 months electrical stimulation, as well as the intactness and thestability; 3) in SD rat retinal homogenate stimulation model, PANi coatingsdramatically protected electrodes and surrounding tissue against nonspecificadsorption and peroxidation, respectively.
PART 3: The fabrication and the investigation of zein conduits in SD rat5mm sciatic nerve regeneration.
The first part was the fabrication and characterization of zein conduits,focus on the conductivity, permeability, bioactivity, mechanical property, etc.The zein conduits and its fabrication were applied China Patent. It indicatedthat 1) zein and its degraded products are biocompatible on PC12 cells; 2) zein conduits were selectively permeable; inner surface coated withnanoscaled microspheres, which was releasable; mechanical property wassuitable for operation and implant; 3) the conductivity of zein conduits wasobtained from PANi coating. PANi are biocompatible on PC12 cells, ratherthan its degraded products, which hamper the cell proliferation in specificcontent; 4) the bioactivity was obtained from implant cells or ECM.The second part was the evaluation of non-bioactive conductive zeinconduits in SD rat model with 5mm sciatic nerve gaps. It showed thatregenerated axons accomplished to connect the distal nerve ends in 4 weeksin both zein conduits and conductive zein conduits. It was feasible inperipheral nerve restoration.
The innovations were mainly in two aspects:
1. Utilization of PANi as coating materials to the implanted electrodes. Itis the first time to evaluate the PANi coating properties in SD rat retinalhomogenate model.
1) The morphology of PANi coating was nanoparticles ranging from20nm to 40nm, which exhibited excellent properties on stability;
2) In long-term electrical stimulation system, the PANi coating showedsplendid anticorrosion property in long-term electrical stimulation, as well asthe intactness and the stability;
3) In the SD rat retinal homogenate electrical stimulation model, thePANi coatings dramatically protected electrodes against nonspecificadsorption, which reduced the inflammation, as well as the protection ofsurrounding tissue from peroxidation.
2. It is firstly to introduce zein into peripheral nerve restoration tofabricate nerve conduits. The zein conduits and its fabrication method were applied China Patent.
1) Zein conduits were biocompatible and biodegradable. PC12 cells relyon the zein degraded products.
2) The procedure of fabrication of zein conduits was simple and wideuse, which could obtain conduits with controllable inner dimmers (>0.28μm),length and depth (>18μm) for tissue engineering.
3) Combined zein with PANi to obtain conductive zein conduits, firsly,for nerve repair and restoration.
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