本文選題：靜電紡絲 + 分級(jí)結(jié)構(gòu)　； 參考：《天津工業(yè)大學(xué)》2016年博士論文

【摘要】：我國(guó)每年由于交通和工業(yè)事故、腫瘤、骨組織壞死和風(fēng)濕等疾病引起的骨組織缺損、骨折病患人數(shù)達(dá)數(shù)百萬(wàn)人,并且隨著社會(huì)人口老齡化的加劇,與骨組織相關(guān)的問(wèn)題越來(lái)越多。當(dāng)前治療骨缺損的方法主要有自體骨、異體骨和人工合成骨移植,其中最有效的治療方法就是自體骨移植,但自體骨移植技術(shù)由于取材來(lái)源有限、功能性障礙、增加患者痛苦等問(wèn)題導(dǎo)致其在臨床應(yīng)用受到了一定限制,而異體骨移植存在價(jià)格昂貴、加工費(fèi)時(shí)等問(wèn)題。因此,設(shè)計(jì)一種在結(jié)構(gòu)、組成和功能上高度模擬天然骨組織基質(zhì),并具備良好的生物相容性和成骨誘導(dǎo)能力的骨修復(fù)材料是當(dāng)前骨組織工程的熱點(diǎn)問(wèn)題。本文圍繞仿生天然骨的分級(jí)結(jié)構(gòu)、組成和功能這一主題,基于納米紡織技術(shù),通過(guò)材料選擇、支架制備、性能表征等方法,在制備出四種不同形式的骨仿生支架并對(duì)這些支架進(jìn)行系統(tǒng)研究的基礎(chǔ)上,探討其在骨組織工程的應(yīng)用潛力。本文主要研究?jī)?nèi)容和結(jié)論如下:首先以仿生天然骨的關(guān)鍵結(jié)構(gòu)特征—礦化的膠原纖維束為目標(biāo),通過(guò)同軸靜電紡絲,制備了以柞蠶絲素蛋白(TSF)為皮層,納米羥基磷灰石(HA)和TSF的復(fù)合粒子為芯層的取向核殼結(jié)構(gòu)納米纖維束。通過(guò)與人骨肉瘤細(xì)胞(MG-63類成骨細(xì)胞)進(jìn)行復(fù)合培養(yǎng)來(lái)評(píng)價(jià)其生物性能。結(jié)果表明,純TSF水溶液顯示出良好的可紡性,在核殼比為1:2和1:1時(shí),皮層的TSF能夠很好的包覆芯層的HA-TSF復(fù)合粒子,特別是在核殼比為1:]的時(shí)候,核殼納米纖維顯示出最佳的力學(xué)性能,其斷裂強(qiáng)度、斷裂伸長(zhǎng)以及初始模量分別為:5.3 MPa,62.6%,70.2 MPa。生物學(xué)性能顯示,與取向的純TSF支架、Cover slip相比,TSF/HA-TSF(1:1)支架展現(xiàn)出最好的細(xì)胞相容性。此外,還發(fā)現(xiàn)纖維的取向?qū)Τ晒羌?xì)胞的排列有重要影響,其生長(zhǎng)方向和纖維的取向是一致的。為了改善支架的力學(xué)性能和生物活性,通過(guò)在聚乳酸-羥基乙酸共聚物(PLGA)中摻雜1wt%氧化石墨烯(GO)納米片和10wt%TSF制備了PLGA/TSF/GO復(fù)合納米纖維支架。結(jié)果表明,GO納米片能夠很好的裹覆和嵌在有機(jī)基質(zhì)(PLGA/TSF)中,在PLGA基質(zhì)中添加TSF和GO后,納米纖維直徑從278 nm減小至130 nm,復(fù)合支架的親水性和蛋白質(zhì)吸附性能得到明顯改善,尤其是力學(xué)性能得到顯著提高,其初始模量和拉伸強(qiáng)度分別是PLGA納米纖維氈的7倍和4倍。生物學(xué)評(píng)價(jià)顯示,PLGA/TSG/GO納米纖維顯示出最高的細(xì)胞增殖能力,而且GO的嵌入和TSF的添加對(duì)功能性地促進(jìn)小鼠骨髓間充質(zhì)干細(xì)胞(mMSCs)分化和新骨的形成起著關(guān)鍵作用。為了獲得在結(jié)構(gòu)、組成上高度仿生并具備良好的力學(xué)性能的骨修復(fù)材料,生物礦化方法已成為實(shí)現(xiàn)形貌結(jié)構(gòu)與天然骨相似的磷灰石在聚合物表面沉積的重要策略。基于以上研究基礎(chǔ),我們制備了 PLGA/TSF/GO三維多層正交的納米纖維支架,并以此為模板通過(guò)模擬體液仿生礦化構(gòu)建了類似于天然骨板層結(jié)構(gòu)的骨修復(fù)材料。結(jié)果顯示,在PLGA納米纖維基質(zhì)中添加TSF能夠誘導(dǎo)HA礦物晶體在納米纖維表面成核和定向生長(zhǎng),TEM結(jié)果顯示纖維表面礦物是由長(zhǎng)40～50 nm,寬2～5 nm的針狀晶體堆砌形成的三維陣列,纖維中存在大量的微孔和介孔結(jié)構(gòu)。礦化后,多層正交結(jié)構(gòu)的PLGA/TSF/GO復(fù)合支架的相比無(wú)紡的PLGA/TSF/GO壓縮模量和壓縮強(qiáng)度分別增加了將近1.7倍和0.6倍。將人骨髓間充質(zhì)干細(xì)胞(hMSCs)接種于復(fù)合支架材料表面,通過(guò)激光共聚焦顯微鏡、ALP、流式細(xì)胞儀分析和實(shí)時(shí)定量PCR等方法檢測(cè)礦化前后纖維支架對(duì)成骨細(xì)胞粘附、增殖、分化的影響,通過(guò)上述實(shí)驗(yàn)證實(shí)了礦化后的納米纖維具有很好的成骨誘導(dǎo)活性,而且細(xì)胞能夠長(zhǎng)入支架內(nèi)部,細(xì)胞排列同樣呈現(xiàn)多層正交的結(jié)構(gòu)。此外,還發(fā)現(xiàn)復(fù)合支架的這種三維多層正交結(jié)構(gòu)也能促進(jìn)細(xì)胞的增殖、分化。為進(jìn)一步對(duì)天然骨的分級(jí)結(jié)構(gòu)進(jìn)行仿生,本文還制備了納米纖維織物為增強(qiáng)的骨仿生復(fù)合材料,這種分級(jí)結(jié)構(gòu)依次為:納米纖維和礦物粒子、礦化的納米纖維、礦化的納米纖維紗、礦化的織物層,直至宏觀結(jié)構(gòu)。首先通過(guò)靜電紡絲技術(shù)制備具有一定捻度的納米纖維紗線,利用紡織編織技術(shù)將納米紗線織成三維多層織物,最后通過(guò)體外細(xì)胞生物礦化在納米纖維表面沉積礦物晶體以構(gòu)建織物增強(qiáng)的納米骨仿生復(fù)合材料。結(jié)果顯示,在聚乳酸(PLA)和TSF質(zhì)量比為9:1時(shí),通過(guò)靜電紡的方法能夠制備連續(xù)的納米紗,且紗線具均勻的細(xì)度和良好的力學(xué)性能。此外,在PLA基質(zhì)中添加TSF能夠顯著改善材料的親水性和蛋白質(zhì)吸附性能。體外細(xì)胞培養(yǎng)結(jié)果顯示,細(xì)胞能夠長(zhǎng)入三維多層納米織物內(nèi)部,并能夠延紗線軸向生長(zhǎng),相比無(wú)紡的納米纖維氈,納米纖維織物中的分級(jí)有序結(jié)構(gòu)能夠更好地促進(jìn)細(xì)胞的增殖、分化和功能性表達(dá)。為了評(píng)價(jià)納米仿生骨材料的促進(jìn)新骨再生能力,動(dòng)物體內(nèi)植入研究發(fā)現(xiàn),我們所制備的體外細(xì)胞礦化后的復(fù)合支架材料具有良好的促進(jìn)新骨再生能力和組織相容性,新形成的骨組織密度為732 ± 56 mg/cm3,接近于天然骨松質(zhì)骨的密度。由此可見,這種具有分級(jí)結(jié)構(gòu)的納米纖維織物骨仿生復(fù)合材料是一種優(yōu)秀的骨替代材料,可用于臨床用于骨缺損治療。
[Abstract]:In our country every year because of traffic and industrial accidents, tumor, bone necrosis and rheumatism and other diseases caused by bone defect, fracture patients the number of millions of people, and with the social population aging, associated with bone tissue more and more problems. The current treatment of bone defects with autogenous bone, allograft bone and synthetic bone graft, which is the most effective method for the treatment of autogenous bone graft with autogenous bone graft, but due to limited sources, functional disorder, increase the suffering of patients and other problems caused by certain restrictions in the clinical application of allograft is expensive, time-consuming and other processing problems. Therefore, in the design of a the structure, composition and function of a high degree of simulation of natural bone matrix, and have the bone repair material compatibility and osteogenic ability good biology is the hot issue in bone tissue engineering. In this paper, a hierarchical structure around the bionic natural bone, the theme of the composition and function of nano textile technology based on, through the selection of materials, scaffold preparation, characterization methods, based on the preparation of four kinds of bone scaffolds of different forms and systematic study of these stents, to explore its potential application in bone tissue engineering. The main research contents and conclusions are as follows: firstly the key structure features of bionic natural bone mineralization of collagen fibers as the goal, through coaxial electrospinning, the tussah silk fibroin was prepared (TSF) to the cortex, nano hydroxyapatite (HA) composite particles and TSF core-shell structure nano fiber orientation as the core layer beam. With human osteosarcoma cells (MG-63 cells) to evaluate its biological properties of composite culture. The results showed that pure TSF aqueous solution showed good spinnability, the core-shell ratio of 1:2 and 1:1 TSF HA-TSF, the cortex can composite particles coated core layer is very good, especially in the core / shell ratio is 1:], core-shell nanofibers showed the best mechanical properties. Its tensile strength, elongation and initial modulus were 5.3 MPa, 62.6% MPa., 70.2 biological properties showed that pure TSF support and orientation of Cover, compared to slip, TSF/HA-TSF (1:1) scaffolds show the best biocompatibility. In addition, also found that the fiber orientation has an important influence on osteoblast arrangement, the growth direction and the fiber orientation is consistent. In order to improve the mechanical properties and biological activity of the frame, through the poly lactic acid glycolic acid (PLGA) in 1wt% doped graphene oxide (GO) PLGA/TSF/GO composite nanofiber scaffolds were fabricated by nano film and 10wt%TSF system. The results show that the nano GO can be well wrapped and embedded in the organic matrix (PLGA/TSF), PLGA matrix The addition of TSF and GO, the nano fiber diameter decreases from 278 nm to 130 nm, the hydrophilicity and protein adsorption properties of the composite scaffold was obviously improved, especially the mechanical properties improved significantly, the initial modulus and tensile strength were 7 times PLGA nanowebs and 4 times. The biological evaluation showed that PLGA/TSG/GO nanofibers showed the highest cell proliferation, and add GO and TSF to the embedded function to promote bone marrow mesenchymal stem cells (mMSCs) plays a key role in the formation and differentiation of new bone. In order to obtain highly bionic structure, composition and possess good mechanical properties of bone repair materials methods, biomineralization has become the important strategy of apatite structure and morphology similar to natural bone deposited on the polymer surface. Based on the research above, we made PLGA/TSF/GO 3D orthogonal multilayer nano fiber preparation Dimensional scaffold, and used as the template by simulated body fluid biomimetic bone repair materials constructed similar to natural bone lamellar structure. The results showed that the addition of TSF can induce HA in mineral crystal nano fiber surface nucleation and directional growth in PLGA nano fiber matrix, TEM results show that the fiber surface is composed of 40~50 nm minerals a three-dimensional array of acicular crystal, 2~5 nm wide pile formation, there are a large number of microporous and mesoporous structure of the fibers. After mineralization, the PLGA/TSF/GO composite support structure compared to the orthogonal multilayer non-woven PLGA/TSF/GO compressive modulus and compressive strength were increased by nearly 1.7 times and 0.6 times. The human bone marrow mesenchymal stem cells (hMSCs) were inoculated on the surface of composite scaffold materials, by laser confocal microscopy, ALP, flow cytometry analysis and real-time PCR method for detection of mineralized fibers before and after stent on osteoblast adhesion, proliferation, The effects of the differentiation through these experiments demonstrated that nano fiber after mineralization has good osteoinductive activity, and the cells could grow into the scaffold structure, arrangement of cells showing the same multilayer orthogonal. In addition, also found that the three-dimensional orthogonal structure composite scaffold can promote cell proliferation and differentiation. For further the hierarchical structure of natural bone biomimetic system, nano fiber fabric for biomimetic composites reinforced by this paper, the hierarchical structure is as follows: nano fibers and nano fiber mineral particles, mineralization, mineralization of nano fiber yarn, fabric layer and mineralization, until the macro structure. Firstly, the nano fiber yarn electrostatic spinning preparation technology with some twist, the textile weaving technology of nano yarn woven into three-dimensional multi-layer fabric, finally through in vitro biomineralization deposition on the surface of nano fiber mineral The crystal structure to construct nano bone biomimetic composite fabric reinforced. The results showed that the polylactic acid (PLA) and TSF mass ratio was 9:1, the preparation of continuous yarn by electrostatic spinning nano method, and yarn with uniform fineness and good mechanical properties. In addition, adding TSF to the hydrophilic and protein adsorption properties of materials significantly improved in the matrix of PLA. Results show the in vitro cell culture, cells can grow into three-dimensional multilayer fabric, and can delay the yarn axial growth, compared to nano fiber non-woven felt, the nanofiber classification in ordered structure can promote cell proliferation, differentiation and function expression. In order to evaluate the nano bionic bone material to promote bone regeneration, implant animal studies found that the composite scaffold materials we prepared in vitro mineralization after preparation can promote new bone regeneration Force and biocompatibility, bone density of newly formed tissue was 732 + 56 mg/cm3, close to natural bone cancellous bone density. Thus, the hierarchical structure with nano fiber fabric bone biomimetic composite is a good bone substitute material, can be used for clinical treatment of bone defects.

【學(xué)位授予單位】：天津工業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R318.08

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