靜電紡可降解高分子超細(xì)纖維在組織工程及藥物釋放中的應(yīng)用研究
發(fā)布時間：2018-10-20 17:43
【摘要】：目前人類健康正面臨著各種各樣的威脅,如疾病、癌癥、意外事故、創(chuàng)傷、外科損傷等等,因此這些問題亟待有效的治療方法。而近年來,組織工程技術(shù)和藥物控制釋放系統(tǒng)越來越受大家的關(guān)注,因?yàn)榻M織工程可以結(jié)合生物學(xué)、醫(yī)學(xué)、材料學(xué)和工程學(xué)等學(xué)科設(shè)計(jì)出可以修復(fù)或者替代受損部位組織工程構(gòu)建物,而藥物控制釋放系統(tǒng)能夠以滿足生理修復(fù)需求、臨床藥物劑量、配方需求的方式按需按時釋放藥物、因子等,因此兩種技術(shù)相結(jié)合,既能相互補(bǔ)充、又能相互促進(jìn)。靜電紡絲是目前常用的制備組織工程支架的方法之一,同時靜電紡絲纖維也是廣泛研究的藥物控制釋放載體之一,所以靜電紡絲技術(shù)能夠結(jié)合兩者長處,在研究領(lǐng)域及臨床應(yīng)用方面具有巨大潛力。本論文旨在研究靜電紡絲技術(shù)在組織工程和藥物控釋方面的結(jié)合應(yīng)用。主要是以生物相容性和可降解性良好的聚乳酸(PLA)、聚ε-己內(nèi)酯(PCL)、聚己內(nèi)酯-聚乙二醇嵌段共聚物(PCL-b-PEG, PCE)等合成高分子為基體材料,制備了復(fù)合磁性納米顆粒的靜電紡絲納米纖維支架,具有控釋功能的載雙藥組織工程支架,以及具有溫度敏感性可控開關(guān)釋藥功能的nanogel-in-fiber器件。對納米顆粒形貌、靜電紡絲纖維的形貌結(jié)構(gòu)、力學(xué)性能、降解性能、藥物控釋能力等進(jìn)行了考察,同時對這些支架進(jìn)行了細(xì)胞學(xué)的評價(jià)或者動物學(xué)評價(jià)。在論文第二章,通過化學(xué)共沉淀法制備了油酸改性超順磁性四氧化三鐵納米粒子(SPIONs),通過動態(tài)光散射儀(DLS)及透射電子顯微鏡(TEM)測量其直徑,通過振動樣品磁強(qiáng)計(jì)(VSM)驗(yàn)證了其超順磁性。將已知固含量的乙醇漿料分散于PLA的DCM/DMF靜電紡絲溶液中用于靜電紡絲并制備了0%-8%(w/w)含量的無規(guī)排列和定向排列的SPIONs/PLA磁性納米纖維。用掃描電子顯微鏡SEM觀察了磁性納米纖維形貌,并通過快速傅立葉變換(FFT)測定了纖維定向度,用萬能力學(xué)試驗(yàn)機(jī)測試了纖維力學(xué)性能。考察了平行于細(xì)胞鋪展平面及垂直于細(xì)胞鋪展平面的磁場和磁性纖維對成骨細(xì)胞單獨(dú)或者共同影響。在論文第三章,通過去溶劑法制備了牛血清白蛋白(BSA)納米顆粒(NPs)及載BMP-2的BSA納米顆粒(BNPs),然后利用靜電自組裝在所制備的納米顆粒上自組裝上殼聚糖外殼以起到穩(wěn)定作用。通過DLS及TEM測量了所制得的納米顆粒直徑。將BNPs與地塞米松(DEX)一起包載于PCE靜電紡絲纖維中,從而制備了可控制釋放的載雙藥組織工程支架,單載DEX, BNPs或NPs的作為對照組,并通過SEM、TEM表征了這些靜電紡絲纖維的形貌及結(jié)構(gòu)。通過紫外可見分光光度計(jì)(UV-Vis)及人BMP-2酶聯(lián)免疫試劑盒分別測量了纖維的載藥效率、載藥量及體外藥物釋曲線。將骨髓來源間充質(zhì)干細(xì)胞(MSCs)接種到載藥PCE纖維支架上后,通過alamarBlue以及活死細(xì)胞染色方法考察了支架的細(xì)胞毒性及細(xì)胞在此纖維支架上鋪展及向內(nèi)遷移等能力,通過21天堿性磷酸酶(ALP)活性測量及第21天的茜素紅染色表征了纖維支架及所載藥物對于MSCs體外誘導(dǎo)成骨能力。在論文第四章,通過體內(nèi)動物實(shí)驗(yàn),考察了載雙藥物PCE纖維支架對于大鼠顱骨8 mm直徑的臨界尺寸缺損的修復(fù)能力。在體外先將MSCs接種到纖維支架上,培養(yǎng)一天后,植入到大鼠顱骨臨界尺寸缺損中。分別于植入4、8和12周后,用X-射線儀觀察顱骨缺損的修復(fù)情況,提取缺損部位剩余材料并用SEM觀察形貌,X射線能譜法(EDX)驗(yàn)證Ca、P鹽沉積以及四氫呋喃(THF)萃取其中高分子成分后測量降解性能。之后切片并進(jìn)行組織化學(xué)染色、免疫組化染色再用光鏡觀察和統(tǒng)計(jì)骨修復(fù)效果,最后討論了BMP-2和DEX促進(jìn)缺損修復(fù)協(xié)同作用的機(jī)理。在論文第五章,考察了不同AAc含量對于共聚溫敏性P(IPAAm-co-AAc)納米凝膠的低臨界溶解溫度(LCST)及敏感性的影響,并成功將P(IPAAm-co-AAc)納米凝膠的LCST上調(diào)到37℃左右。通過UV-Vis測量了LCST,通過DLS測量了響應(yīng)性納米凝膠的粒徑隨溫度的變化。通過靜電紡絲技術(shù)將P(IPAAm-co-AAc)納米凝膠載于PCL/PEO核殼纖維的PCL殼層作為可溫敏性開關(guān)藥物釋放的開關(guān),而甲基橙作為藥物模板及顏色指示劑攜載于PEO核層,得到了可溫敏性開關(guān)藥物釋放的nanogel-in-microfiber器件,之后通過SEM觀察了纖維的形貌,TEM驗(yàn)證了載納米凝膠的纖維結(jié)構(gòu),然后在體外考察了此核殼纖維響應(yīng)性藥物釋放特性。此外還制備了載鹽酸阿霉素(DOX)的溫敏性核殼纖維,通過體外對小鼠乳腺癌4T1細(xì)胞的抑制實(shí)驗(yàn)說明了這種響應(yīng)性核殼纖維的潛在應(yīng)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，
本文編號：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，
本文編號：2283915
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