担载神经生长因子(NGF)的乳液法涂层纤维体外缓释研究

目的:目前周围神经修复中,神经导管是研究热点,本文研究乳液法涂层纤维制备的神经导管在神经修复中应用的可能性。方法:本文采用乳液法制备担载NGF的丝素-聚乳酸(PLLA)涂层电纺纤维,观察纤维的形态,测定NGF的体外释放动力学参数,并考察纤维释放液对于PC12细胞增殖的影响。结果:担载NGF的涂层纤维具备类似于细胞外基质(ECM)的三维结构和多孔形态;涂层纤维中NGF体外有效缓释10天;细胞实验中,在含有释放液的培养基中生长的PC12细胞,与空白对照组相比,荧光强度平均多了2000-4000个荧光强度,所以释放液可以更好地促进PC12细胞的增殖。结论:担载NGF的乳液法涂层纺丝纤维具备促进缺损周围神经修复的条件,可以进一步研究在动物体内修复缺损周围神经中的效果,为以后的临床应用打下基础。     

NGF电纺纤维的体外缓释研究

目的:研究担载神经生长因子(NGF)的静电纺丝纤维的表征,考察NGF电纺纤维对于周围神经修复的效果。方法:将NGF水溶液分散于PLLA溶液,通过W/O乳液法制备静电纺丝纤维,对纤维的形态、力学性能等进行表征,Elisa方法测定NGF的体外释放动力学,Alamer Blue法检测试剂来考察纤维释放液对于PC12细胞增殖的影响。结果:NGF电纺纤维具备良好的形态和力学性质,直径为500-900 nm,纤维具备三维多孔结构。纤维的最大拉伸应力为2.50±0.41 MPa。电纺纤维中NGF在体外可有效释放9天,累积释放量接近3000 pg。细胞活性实验结果显示,第1、3、5、7天释放液的荧光强度与对照组相比有显著差异。结论:担载NGF的乳液法静电纺丝纤维有促进缺损周围神经修复的潜质。     

血管内皮生长因子(VEGF)乳液法电纺纤维膜的体外研究

目的:研究担载血管内皮生长因子(VEGF)的乳液法电纺纤维膜的亲水性能、外观形态和机械性能,纤维膜中VEGF的包封率和体外释放动力学,为评价其能否应用于血管再生领域的研究奠定基础。方法:将VEGF水溶液通过W/O乳液法制备成缓释VEGF的生物可降解的丙交酯-乙交酯共聚物(PLGA)静电纺丝纤维膜,对该纤维膜的接触角、外观形态、机械性能进行表征,Elisa法测定该纤维膜的体外14天的释放行为,分别观察纤维膜释放0天、7天、14天后的电镜图。结果:加入VEGF后,纤维膜的接触角由140.0°减小到136.1°,亲水性增强,具有类似细胞外基质(ECMs)网状结构和良好的力学性能,纤维膜第1天的突释不超过载药量的50%,电镜图下显示纤维膜释放1周时纤维发生断裂。结论:通过乳液法制备的担载VEGF的电纺纤维膜具有良好的物理性能,能够持续缓释VEGF,可作为血管再生的组织工程支架进行深入研究。     

静电纺丝制备担载神经生长因子的聚乳酸纤维的工艺研究

目的:研究担载神经生长因子(NGF)的聚乳酸纤维乳液法静电纺丝的制备工艺,从电压、溶液浓度等工艺条件进行探索,通过扫描电镜对纤维的形态结构进行观察,旨在找到最佳纺丝制备条件,并观察该条件下纤维的体外释放行为和细胞活性。方法:将NGF水溶液分散于聚乳酸(PLLA)溶液中,通过W/O乳液法制备静电纺丝纤维。分别从电压8 k V、10 k V、12 k V,浓度梯度90mg/m L、100 mg/m L、110 mg/m L进行静电纺丝纤维的制备,对纤维的形态等进行表征。使用ELISA对NGF体外释放动力学进行检测,用Alamer Blue试剂考察纤维释放液对于PC12悬浮细胞增殖的影响。结果:浓度和电压对电纺纤维制备影响很大。当浓度过大时,易堵塞纺丝喷头且纤维弯曲,过小时纤维粗细差异较大。电压过大或过小时纤维弯曲情况严重,甚至出现缠绕现象。当浓度为100 mg/m L,电压为10 k V时制备的乳液法静电纺丝聚乳酸纤维直径粗细均匀,具有较好形态。在该条件下的制备的纤维NGF体外有效释放13天,释放液可以促进PC12细胞的增殖。结论:担载NGF的聚乳酸纤维乳液法最佳静电纺丝制备条件为:PLLA溶液浓度100 mg/m L、电压10 k V,该条件下制备的担载NGF的聚乳酸纤维体外释放可累计释放13天,其释放液可有效促进PC12细胞的增殖,为进一步研究担载NGF的聚乳酸纤维导管奠定了一定的工艺基础。     

担载血管生长因子的乳液法纤维用于血管再生的研究

目的:制备担载血管生长因子(VEGF)的乳液法静电纺丝纤维膜,对其开展一系列表征,从而研究其血管再生的潜能。方法:通过W/O乳液法制备担载VEGF的静电纺丝纤维膜,并对其形态、力学性质进行表征。用VEGF ELISA分析方法对其体外释放动力学进行研究。运用CCK-8法检测乳液法静电纺丝纤维膜中VEGF的活性变化。结果:乳液法静电纺丝纤维膜呈现连通的三维网状结构,平均直径为1μm,模拟了细胞外基质(ECM),最大拉伸应力为3.03±0.66 M Pa,具有良好的抗拉伸能力,能够支持细胞的生长。乳液法纤维膜中VEGF在体外累积释放了14天,总释放量超过20000 pg,达到血管再生的有效浓度。CCK-8结果显示,乳液法纤维膜中的VEGF仍然保持较高的蛋白活性。结论:担载VEGF的乳液法静电纺丝纤维膜能够缓释出活性的蛋白,具有血管再生的潜能。     

可控性释放NGF的京尼平- 壳聚糖微球的研究

目的:在体外研究京尼平-壳聚糖微球可控性释放具有生物活性的神经生长因子的可行性。方法:采用"乳化-化学交联"技术制备包埋神经生长因子的京尼平-壳聚糖微球,京尼平为化学交联剂;应用扫描电镜、粒径分布、体外缓释动力学及细胞生物活性分别对微球的性能进行研究。结果:京尼平-壳聚糖微球表面光滑,平均粒径在5.1~50.5μm之间;京尼平的浓度可影响微球在体外释放神经生长因子的速度,经高浓度京尼平交联的微球能减缓并持续释放神经生长因子;此外,从京尼平-壳聚糖微球释放的神经生长因子可维持PC12细胞的生物活性,提高NGF生物利用率。结论:京尼平-壳聚糖微球能有效缓释具有生物活性的NGF超过14天,为神经退行性疾病的治疗提供一种治疗策略。     

聚乙烯醇复合水凝胶外用膜剂的制备与性能

目的：考察PVA／葡聚糖／羧甲基纤维素钠复合水凝胶外用膜剂的制备方法,并与纯PVA水凝胶贴膜进行对比,考察本膜剂在物理性能和药物体系的体外释放行为上所具备的优越性。方法：利用冷冻-解冻物理交联方法制备水凝胶装载胰岛素模型药物的外用膜剂,通过万能拉力机和差示扫描量热法考察膜剂的物理性能,利用高效液色谱法考察该膜剂的体外释放行为。结果：PVA复合水凝胶外用贴膜相较于纯PVA水凝胶贴膜的韧性减小、刚性增加,体外释放变好。结论：通过将具有材料友好性的PVA和多糖葡聚糖、羧甲基纤维素钠合并使用制备胰岛素复合水凝胶贴膜,既能保证贴膜具有良好的物理性能,又具有较好地释放行为,优于目前文献报道的纯PVA水凝胶贴膜性能,有望继续研究优化性能。     

聚乙烯醇复合水凝胶外用膜剂的制备与性能*

摘要目的：考察PVA/ 葡聚糖/ 羧甲基纤维素钠复合水凝胶外用膜剂的制备方法,并与纯PVA 水凝胶贴膜进行对比,考察本膜 剂在物理性能和药物体系的体外释放行为上所具备的优越性。方法：利用冷冻-解冻物理交联方法制备水凝胶装载胰岛素模型药 物的外用膜剂,通过万能拉力机和差示扫描量热法考察膜剂的物理性能,利用高效液色谱法考察该膜剂的体外释放行为。结果： PVA复合水凝胶外用贴膜相较于纯PVA水凝胶贴膜的韧性减小、刚性增加,体外释放变好。结论：通过将具有材料友好性的PVA 和多糖葡聚糖、羧甲基纤维素钠合并使用制备胰岛素复合水凝胶贴膜,既能保证贴膜具有良好的物理性能,又具有较好地释放行 为,优于目前文献报道的纯PVA水凝胶贴膜性能,有望继续研究优化性能。     

乳液法静电纺丝PLGA组织工程缓释纤维的研究

目的：研究Dextran对蛋白药物的释放影响。方法：将模型蛋白BSA溶解于多糖溶液中,通过W/O乳液法静电纺丝制备缓释纤维。采用MicroBCA法测定该纤维体外释放行为,采用SEC-HPLC检测制备前后蛋白的聚集程度,并与不含多糖的BSA纤维做对照。结果：添加Dextran以后蛋白的包封率由52.68%提高到63.92%,第一天突释不大于药物载量的15%,对蛋白单体的保持达到85%以上。结论：Dextran可以改善一般组织工程纤维中蛋白药物的释放,提高蛋白药物在制剂、贮存、释放过程中的稳定性,增加纤维的载药量。     

装载于PLGA中的5-氟尿嘧啶缓释微球的制备及其体外释放的研究

目的：研究装载于不同分子量的PLGA中的5-氟尿嘧啶微球的制备方法及其在体外条件下的缓释行为。方法：以水包油包固复乳法将5-氟尿嘧啶包裹在高分子聚乳酸-聚羟基乙酸共聚物（PLGA）中,形成缓释微球,考察其大小,外观,包封率等理化性质,以紫外分光光度法为检测方法研究其体外释放行为。结果：经扫描电子显微镜观察,所制备的微球形态完整,大小较均匀。具有一定得包封率和载药量,体外释放研究表明其处方1和处方2的缓释时间为8天和23天。结论：以水包油包固复乳法制备的PLGA 5-氟尿嘧啶微球能够达到缓释的目的。     

Complementary Effects of Two Growth Factors in Multifunctionalized Silk Nanofibers for Nerve Reconstruction

With the aim of forming bioactive guides for peripheral nerve regeneration, silk fibroin was electrospun to obtain aligned nanofibers. These fibers were functionalized by incorporating Nerve Growth Factor (NGF) and Ciliary NeuroTrophic Factor (CNTF) during electrospinning. PC12 cells grown on the fibers confirmed the bioavailability and bioactivity of the NGF, which was not significantly released from the fibers. Primary neurons from rat dorsal root ganglia (DRGs) were grown on the nanofibers and anchored to the fibers and grew in a directional fashion based on the fiber orientation, and as confirmed by growth cone morphology. These biofunctionalized nanofibers led to a 3-fold increase in neurite length at their contact, which was likely due to the NGF. Glial cell growth, alignment and migration were stimulated by the CNTF in the functionalized nanofibers. Organotypic culture of rat fetal DRGs confirmed the complementary effect of both growth factors in multifunctionalized nanofibers, which allowed glial cell migration, alignment and parallel axonal growth in structures resembling the ‘bands of Bungner’ found in situ. Graftable multi-channel conduits based on biofunctionalized aligned silk nanofibers were developed as an organized 3D scaffold. Our bioactive silk tubes thus represent new options for a biological and biocompatible nerve guidance conduit.     

PLGA/ECM神经支架性质的体外评价

以赖氨酸、神经生长因子(NGF)、聚乳酸聚羟基乙酸共聚物(PLGA)、猪皮来源的细胞外基质(ECM)为原料制备了一种复合材料;考察其内部三维结构,生物力学性质,降解特性,雪旺氏细胞黏附状况,以及其对NGF的可控释放作用;从而评价其作为促周围神经损伤修复支架的可行性。扫描电子显微镜(SEM)观察显示,PLGA渗透入去细胞猪皮内部固有的蜂窝状孔隙中,并覆盖在孔隙内表面;孔隙率为68.3%～81.2%,密度为0.62～0.68 g/cm3。复合材料的断裂强度为8.308 MPa,断裂伸长率为38.98%,弹性模量为97.27 MPa;在4周的体外降解测试中,其最大失重率为43.3%;赖氨酸在复合材料中的添加对降解液pH的相对稳定具有显著作用;在30 d中,复合材料对NGF的累积释放率为38%;通过雪旺氏细胞与复合材料的共培养,发现雪旺氏细胞能够在其表面及孔隙中黏附。因此表明本复合材料有望成为一种新型的促周围神经损伤修复支架。     

Sustained release of proteins from electrospun biodegradable fibers

Electrospinning is a simple and versatile technique of producing polymeric fibers ranging from submicron to micron in diameter. Incorporation of bioactive agents into the fibers could make a biofunctional tissue engineering scaffold. In this study, we investigated the feasibility of encapsulating human beta-nerve growth factor (NGF), which was stabilized in a carrier protein, bovine serum albumin (BSA) in a copolymer of epsilon-caprolactone and ethyl ethylene phosphate (PCLEEP) by electrospinning. Partially aligned protein encapsulated fibers were obtained and the protein was found to be randomly dispersed throughout the electrospun fibrous mesh in aggregate form. A sustained release of NGF via diffusion process was obtained for at least 3 months. PC12 neurite outgrowth assay confirmed that the bioactivity of electrospun NGF was retained, at least partially, throughout the period of sustained release, thus clearly demonstrating the feasibility of encapsulating proteins via electrospinning to produce biofunctional tissue scaffolds.     

Endogenous chicken nerve growth factor from sheath cells is transported in regenerating nerve

We report the presence of endogenous nerve growth factor (NGF) in chicken peripheral nerve. The molecule has been detected with antibodies to mouse salivary gland NGF, using immunohistochemical and immunoelectrophoretic techniques. Previous studies have shown that these antibodies inhibit the survival activity of extracts of chicken peripheral nerve. The NGF accumulated distal, but not proximal, to a ligature placed on a peripheral sympathetic nerve demonstrating that it was retrogradely transported. This transport was detected in intact nerve fibers as well as in nerves from which the peripheral target had been ablated 6 hr or 7 days previously. The results indicate that avian NGF is present in adult chicken peripheral nerves and that this molecule shares antigenic determinants with the mouse molecule. The results further demonstrate that regenerating neurons retrogradely transport NGF supplied by cells within the peripheral nerve (presumably Schwann). The possibility that these cells also provide NGF to intact neurons is discussed.     

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.