静电纺可降解高分子超细纤维在组织工程及药物释放中的应用研究
发布时间：2018-10-20 17:43
【摘要】：目前人类健康正面临着各种各样的威胁,如疾病、癌症、意外事故、创伤、外科损伤等等,因此这些问题亟待有效的治疗方法。而近年来,组织工程技术和药物控制释放系统越来越受大家的关注,因为组织工程可以结合生物学、医学、材料学和工程学等学科设计出可以修复或者替代受损部位组织工程构建物,而药物控制释放系统能够以满足生理修复需求、临床药物剂量、配方需求的方式按需按时释放药物、因子等,因此两种技术相结合,既能相互补充、又能相互促进。静电纺丝是目前常用的制备组织工程支架的方法之一,同时静电纺丝纤维也是广泛研究的药物控制释放载体之一,所以静电纺丝技术能够结合两者长处,在研究领域及临床应用方面具有巨大潜力。本论文旨在研究静电纺丝技术在组织工程和药物控释方面的结合应用。主要是以生物相容性和可降解性良好的聚乳酸(PLA)、聚ε-己内酯(PCL)、聚己内酯-聚乙二醇嵌段共聚物(PCL-b-PEG, PCE)等合成高分子为基体材料,制备了复合磁性纳米颗粒的静电纺丝纳米纤维支架,具有控释功能的载双药组织工程支架,以及具有温度敏感性可控开关释药功能的nanogel-in-fiber器件。对纳米颗粒形貌、静电纺丝纤维的形貌结构、力学性能、降解性能、药物控释能力等进行了考察,同时对这些支架进行了细胞学的评价或者动物学评价。在论文第二章,通过化学共沉淀法制备了油酸改性超顺磁性四氧化三铁纳米粒子(SPIONs),通过动态光散射仪(DLS)及透射电子显微镜(TEM)测量其直径,通过振动样品磁强计(VSM)验证了其超顺磁性。将已知固含量的乙醇浆料分散于PLA的DCM/DMF静电纺丝溶液中用于静电纺丝并制备了0%-8%(w/w)含量的无规排列和定向排列的SPIONs/PLA磁性纳米纤维。用扫描电子显微镜SEM观察了磁性纳米纤维形貌,并通过快速傅立叶变换(FFT)测定了纤维定向度,用万能力学试验机测试了纤维力学性能。考察了平行于细胞铺展平面及垂直于细胞铺展平面的磁场和磁性纤维对成骨细胞单独或者共同影响。在论文第三章,通过去溶剂法制备了牛血清白蛋白(BSA)纳米颗粒(NPs)及载BMP-2的BSA纳米颗粒(BNPs),然后利用静电自组装在所制备的纳米颗粒上自组装上壳聚糖外壳以起到稳定作用。通过DLS及TEM测量了所制得的纳米颗粒直径。将BNPs与地塞米松(DEX)一起包载于PCE静电纺丝纤维中,从而制备了可控制释放的载双药组织工程支架,单载DEX, BNPs或NPs的作为对照组,并通过SEM、TEM表征了这些静电纺丝纤维的形貌及结构。通过紫外可见分光光度计(UV-Vis)及人BMP-2酶联免疫试剂盒分别测量了纤维的载药效率、载药量及体外药物释曲线。将骨髓来源间充质干细胞(MSCs)接种到载药PCE纤维支架上后,通过alamarBlue以及活死细胞染色方法考察了支架的细胞毒性及细胞在此纤维支架上铺展及向内迁移等能力,通过21天碱性磷酸酶(ALP)活性测量及第21天的茜素红染色表征了纤维支架及所载药物对于MSCs体外诱导成骨能力。在论文第四章,通过体内动物实验,考察了载双药物PCE纤维支架对于大鼠颅骨8 mm直径的临界尺寸缺损的修复能力。在体外先将MSCs接种到纤维支架上,培养一天后,植入到大鼠颅骨临界尺寸缺损中。分别于植入4、8和12周后,用X-射线仪观察颅骨缺损的修复情况,提取缺损部位剩余材料并用SEM观察形貌,X射线能谱法(EDX)验证Ca、P盐沉积以及四氢呋喃(THF)萃取其中高分子成分后测量降解性能。之后切片并进行组织化学染色、免疫组化染色再用光镜观察和统计骨修复效果,最后讨论了BMP-2和DEX促进缺损修复协同作用的机理。在论文第五章,考察了不同AAc含量对于共聚温敏性P(IPAAm-co-AAc)纳米凝胶的低临界溶解温度(LCST)及敏感性的影响,并成功将P(IPAAm-co-AAc)纳米凝胶的LCST上调到37℃左右。通过UV-Vis测量了LCST,通过DLS测量了响应性纳米凝胶的粒径随温度的变化。通过静电纺丝技术将P(IPAAm-co-AAc)纳米凝胶载于PCL/PEO核壳纤维的PCL壳层作为可温敏性开关药物释放的开关,而甲基橙作为药物模板及颜色指示剂携载于PEO核层,得到了可温敏性开关药物释放的nanogel-in-microfiber器件,之后通过SEM观察了纤维的形貌,TEM验证了载纳米凝胶的纤维结构,然后在体外考察了此核壳纤维响应性药物释放特性。此外还制备了载盐酸阿霉素(DOX)的温敏性核壳纤维,通过体外对小鼠乳腺癌4T1细胞的抑制实验说明了这种响应性核壳纤维的潜在应用。
[Abstract]:Human health is facing a wide variety of threats, such as disease, cancer, accidents, trauma, surgical injuries, and so on, so these problems need to be effectively addressed. In recent years, tissue engineering and drug control release systems are becoming more and more concerned, as tissue engineering can be designed to repair or replace damaged site tissue engineering constructs in conjunction with disciplines such as biology, medicine, material science and engineering, and the drug control release system can release drugs, factors and the like on time according to the requirements of physiological repair requirements, clinical drug dosage and formula requirements, so that the two technologies can supplement each other and promote each other. Electrostatic spinning is one of the commonly used methods for preparing tissue engineering scaffold, while electrostatic spinning fiber is one of the widely studied drug control release carriers, so the electrostatic spinning technology can combine the advantages of both, and has great potential in research field and clinical application. The purpose of this paper is to study the application of electrostatic spinning technology in tissue engineering and drug controlled release. The electrostatic spinning nanofiber scaffold is prepared from polylactic acid (PLA), poly (lactide-hexyl) lactone (PCL), polyhexolide-polyethylene glycol block copolymer (PCL-b-PEG, PCE) and the like with good biocompatibility and degradability, The invention relates to a double-drug tissue engineering scaffold with a controlled-release function and an ogel-in-fiber device with a temperature-sensitive controllable switch-releasing function. The morphology of nano-particles, morphology, mechanical properties, degradation properties and controlled-release ability of electrostatic spinning fiber were investigated, and cytological evaluation or zoology evaluation was carried out on these scaffolds. In the second chapter of the thesis, oleic acid modified superparamagnetic ferroferric oxide nanoparticles (SPIONs) were prepared by chemical co-precipitation method. The diameters of the superparamagnetic ferroferric oxide nanoparticles (SPIONs) were measured by dynamic light scattering instrument (DLS) and transmission electron microscopy (TEM). The superparamagnetism was verified by shaking sample magnetometer (VSM). A known solid content of ethanol slurry was dispersed in a PLA DCM/ DMF electrostatic spinning solution for electrostatic spinning and a random array of 0% to 8% (w/ w) content of SPIONs/ PLA magnetic nanofibers was prepared. The morphology of the magnetic nano-fiber was observed by scanning electron microscope (SEM), and the fiber orientation was determined by FFT. The mechanical properties of the fiber were tested with a universal mechanical testing machine. The effects of magnetic field and magnetic fibers, which are parallel to the cell spreading plane and perpendicular to the cell spreading plane, on osteoblasts were investigated either alone or in common. In chapter 3 of the thesis, bovine serum albumin (BSA) nanoparticles (NPs) and BSA nanoparticles carrying BMP-2 were prepared by de-solvent method, and then the chitosan shell was self-assembled on the prepared nanoparticles by electrostatic self-assembly to play a stable role. The prepared nanoparticles were measured by DLS and TEM. LPs and dexamethasone (DEX) were packaged in PCE electrostatic spinning fiber, so as to prepare the controlled release drug-carrying tissue engineering scaffold, single-carrier DEX, PSPs or NPs as control group, and the morphology and structure of these electrostatic spinning fibers were characterized by SEM and TEM. The drug loading efficiency, drug loading rate and drug release curve in vitro were measured by UV-Vis and human BMP-2 enzyme-linked immunosorbent assay. After the bone marrow-derived mesenchymal stem cells (MSCs) were inoculated onto a drug-loaded PCE fiber scaffold, the cell toxicity of the scaffold and the ability of the cells to spread and migrate inward on the scaffold were investigated by alamarBlue and dead cell staining. Through 21 days alkaline phosphatase (ALP) activity measurement and 21 days red staining, the fiber scaffold and the drug loaded were characterized by the ability of MSCs to induce bone in vitro. In the fourth chapter of the thesis, through in vivo animal experiment, the repair ability of the double drug PCE fiber stent to the critical dimension defect of the 8 mm diameter of the skull of the rat was investigated. MSCs were seeded onto the fiber scaffold in vitro and then implanted into the critical size defect of the rat skull after one day. After 4, 8 and 12 weeks respectively, the repair of the skull defect was observed with an X-ray apparatus, the remaining materials were extracted and the morphology was observed by SEM, and the properties of degradation were measured by X-ray spectroscopy (EDX), Ca, P salt deposition and THF extraction. The effect of BMP-2 and DEX on the synergistic effect of BMP-2 and DEX were discussed. In the fifth chapter, the effect of different AAc contents on the low critical dissolution temperature (LCST) and sensitivity of the copolymerization temperature sensitive P (IPAAm-co-AAc) nano-gel was investigated, and the LCST of P (IPAAm-co-AAc) nano-gel was successfully raised to about 37 鈩，
本文编号：2283915
[Abstract]:Human health is facing a wide variety of threats, such as disease, cancer, accidents, trauma, surgical injuries, and so on, so these problems need to be effectively addressed. In recent years, tissue engineering and drug control release systems are becoming more and more concerned, as tissue engineering can be designed to repair or replace damaged site tissue engineering constructs in conjunction with disciplines such as biology, medicine, material science and engineering, and the drug control release system can release drugs, factors and the like on time according to the requirements of physiological repair requirements, clinical drug dosage and formula requirements, so that the two technologies can supplement each other and promote each other. Electrostatic spinning is one of the commonly used methods for preparing tissue engineering scaffold, while electrostatic spinning fiber is one of the widely studied drug control release carriers, so the electrostatic spinning technology can combine the advantages of both, and has great potential in research field and clinical application. The purpose of this paper is to study the application of electrostatic spinning technology in tissue engineering and drug controlled release. The electrostatic spinning nanofiber scaffold is prepared from polylactic acid (PLA), poly (lactide-hexyl) lactone (PCL), polyhexolide-polyethylene glycol block copolymer (PCL-b-PEG, PCE) and the like with good biocompatibility and degradability, The invention relates to a double-drug tissue engineering scaffold with a controlled-release function and an ogel-in-fiber device with a temperature-sensitive controllable switch-releasing function. The morphology of nano-particles, morphology, mechanical properties, degradation properties and controlled-release ability of electrostatic spinning fiber were investigated, and cytological evaluation or zoology evaluation was carried out on these scaffolds. In the second chapter of the thesis, oleic acid modified superparamagnetic ferroferric oxide nanoparticles (SPIONs) were prepared by chemical co-precipitation method. The diameters of the superparamagnetic ferroferric oxide nanoparticles (SPIONs) were measured by dynamic light scattering instrument (DLS) and transmission electron microscopy (TEM). The superparamagnetism was verified by shaking sample magnetometer (VSM). A known solid content of ethanol slurry was dispersed in a PLA DCM/ DMF electrostatic spinning solution for electrostatic spinning and a random array of 0% to 8% (w/ w) content of SPIONs/ PLA magnetic nanofibers was prepared. The morphology of the magnetic nano-fiber was observed by scanning electron microscope (SEM), and the fiber orientation was determined by FFT. The mechanical properties of the fiber were tested with a universal mechanical testing machine. The effects of magnetic field and magnetic fibers, which are parallel to the cell spreading plane and perpendicular to the cell spreading plane, on osteoblasts were investigated either alone or in common. In chapter 3 of the thesis, bovine serum albumin (BSA) nanoparticles (NPs) and BSA nanoparticles carrying BMP-2 were prepared by de-solvent method, and then the chitosan shell was self-assembled on the prepared nanoparticles by electrostatic self-assembly to play a stable role. The prepared nanoparticles were measured by DLS and TEM. LPs and dexamethasone (DEX) were packaged in PCE electrostatic spinning fiber, so as to prepare the controlled release drug-carrying tissue engineering scaffold, single-carrier DEX, PSPs or NPs as control group, and the morphology and structure of these electrostatic spinning fibers were characterized by SEM and TEM. The drug loading efficiency, drug loading rate and drug release curve in vitro were measured by UV-Vis and human BMP-2 enzyme-linked immunosorbent assay. After the bone marrow-derived mesenchymal stem cells (MSCs) were inoculated onto a drug-loaded PCE fiber scaffold, the cell toxicity of the scaffold and the ability of the cells to spread and migrate inward on the scaffold were investigated by alamarBlue and dead cell staining. Through 21 days alkaline phosphatase (ALP) activity measurement and 21 days red staining, the fiber scaffold and the drug loaded were characterized by the ability of MSCs to induce bone in vitro. In the fourth chapter of the thesis, through in vivo animal experiment, the repair ability of the double drug PCE fiber stent to the critical dimension defect of the 8 mm diameter of the skull of the rat was investigated. MSCs were seeded onto the fiber scaffold in vitro and then implanted into the critical size defect of the rat skull after one day. After 4, 8 and 12 weeks respectively, the repair of the skull defect was observed with an X-ray apparatus, the remaining materials were extracted and the morphology was observed by SEM, and the properties of degradation were measured by X-ray spectroscopy (EDX), Ca, P salt deposition and THF extraction. The effect of BMP-2 and DEX on the synergistic effect of BMP-2 and DEX were discussed. In the fifth chapter, the effect of different AAc contents on the low critical dissolution temperature (LCST) and sensitivity of the copolymerization temperature sensitive P (IPAAm-co-AAc) nano-gel was investigated, and the LCST of P (IPAAm-co-AAc) nano-gel was successfully raised to about 37 鈩，
本文编号：2283915
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