丝素纳米纤维骨仿生复合材料的构建及应用

发布时间：2018-04-13 06:04

本文选题：静电纺丝 + 分级结构　；参考：《天津工业大学》2016年博士论文

【摘要】：我国每年由于交通和工业事故、肿瘤、骨组织坏死和风湿等疾病引起的骨组织缺损、骨折病患人数达数百万人,并且随着社会人口老龄化的加剧,与骨组织相关的问题越来越多。当前治疗骨缺损的方法主要有自体骨、异体骨和人工合成骨移植,其中最有效的治疗方法就是自体骨移植,但自体骨移植技术由于取材来源有限、功能性障碍、增加患者痛苦等问题导致其在临床应用受到了一定限制,而异体骨移植存在价格昂贵、加工费时等问题。因此,设计一种在结构、组成和功能上高度模拟天然骨组织基质,并具备良好的生物相容性和成骨诱导能力的骨修复材料是当前骨组织工程的热点问题。本文围绕仿生天然骨的分级结构、组成和功能这一主题,基于纳米纺织技术,通过材料选择、支架制备、性能表征等方法,在制备出四种不同形式的骨仿生支架并对这些支架进行系统研究的基础上,探讨其在骨组织工程的应用潜力。本文主要研究内容和结论如下:首先以仿生天然骨的关键结构特征—矿化的胶原纤维束为目标,通过同轴静电纺丝,制备了以柞蚕丝素蛋白(TSF)为皮层,纳米羟基磷灰石(HA)和TSF的复合粒子为芯层的取向核壳结构纳米纤维束。通过与人骨肉瘤细胞(MG-63类成骨细胞)进行复合培养来评价其生物性能。结果表明,纯TSF水溶液显示出良好的可纺性,在核壳比为1:2和1:1时,皮层的TSF能够很好的包覆芯层的HA-TSF复合粒子,特别是在核壳比为1:]的时候,核壳纳米纤维显示出最佳的力学性能,其断裂强度、断裂伸长以及初始模量分别为:5.3 MPa,62.6%,70.2 MPa。生物学性能显示,与取向的纯TSF支架、Cover slip相比,TSF/HA-TSF(1:1)支架展现出最好的细胞相容性。此外,还发现纤维的取向对成骨细胞的排列有重要影响,其生长方向和纤维的取向是一致的。为了改善支架的力学性能和生物活性,通过在聚乳酸-羟基乙酸共聚物(PLGA)中掺杂1wt%氧化石墨烯(GO)纳米片和10wt%TSF制备了PLGA/TSF/GO复合纳米纤维支架。结果表明,GO纳米片能够很好的裹覆和嵌在有机基质(PLGA/TSF)中,在PLGA基质中添加TSF和GO后,纳米纤维直径从278 nm减小至130 nm,复合支架的亲水性和蛋白质吸附性能得到明显改善,尤其是力学性能得到显著提高,其初始模量和拉伸强度分别是PLGA纳米纤维毡的7倍和4倍。生物学评价显示,PLGA/TSG/GO纳米纤维显示出最高的细胞增殖能力,而且GO的嵌入和TSF的添加对功能性地促进小鼠骨髓间充质干细胞(mMSCs)分化和新骨的形成起着关键作用。为了获得在结构、组成上高度仿生并具备良好的力学性能的骨修复材料,生物矿化方法已成为实现形貌结构与天然骨相似的磷灰石在聚合物表面沉积的重要策略。基于以上研究基础,我们制备了 PLGA/TSF/GO三维多层正交的纳米纤维支架,并以此为模板通过模拟体液仿生矿化构建了类似于天然骨板层结构的骨修复材料。结果显示,在PLGA纳米纤维基质中添加TSF能够诱导HA矿物晶体在纳米纤维表面成核和定向生长,TEM结果显示纤维表面矿物是由长40～50 nm,宽2～5 nm的针状晶体堆砌形成的三维阵列,纤维中存在大量的微孔和介孔结构。矿化后,多层正交结构的PLGA/TSF/GO复合支架的相比无纺的PLGA/TSF/GO压缩模量和压缩强度分别增加了将近1.7倍和0.6倍。将人骨髓间充质干细胞(hMSCs)接种于复合支架材料表面,通过激光共聚焦显微镜、ALP、流式细胞仪分析和实时定量PCR等方法检测矿化前后纤维支架对成骨细胞粘附、增殖、分化的影响,通过上述实验证实了矿化后的纳米纤维具有很好的成骨诱导活性,而且细胞能够长入支架内部,细胞排列同样呈现多层正交的结构。此外,还发现复合支架的这种三维多层正交结构也能促进细胞的增殖、分化。为进一步对天然骨的分级结构进行仿生,本文还制备了纳米纤维织物为增强的骨仿生复合材料,这种分级结构依次为:纳米纤维和矿物粒子、矿化的纳米纤维、矿化的纳米纤维纱、矿化的织物层,直至宏观结构。首先通过静电纺丝技术制备具有一定捻度的纳米纤维纱线,利用纺织编织技术将纳米纱线织成三维多层织物,最后通过体外细胞生物矿化在纳米纤维表面沉积矿物晶体以构建织物增强的纳米骨仿生复合材料。结果显示,在聚乳酸(PLA)和TSF质量比为9:1时,通过静电纺的方法能够制备连续的纳米纱,且纱线具均匀的细度和良好的力学性能。此外,在PLA基质中添加TSF能够显著改善材料的亲水性和蛋白质吸附性能。体外细胞培养结果显示,细胞能够长入三维多层纳米织物内部,并能够延纱线轴向生长,相比无纺的纳米纤维毡,纳米纤维织物中的分级有序结构能够更好地促进细胞的增殖、分化和功能性表达。为了评价纳米仿生骨材料的促进新骨再生能力,动物体内植入研究发现,我们所制备的体外细胞矿化后的复合支架材料具有良好的促进新骨再生能力和组织相容性,新形成的骨组织密度为732 ± 56 mg/cm3,接近于天然骨松质骨的密度。由此可见,这种具有分级结构的纳米纤维织物骨仿生复合材料是一种优秀的骨替代材料,可用于临床用于骨缺损治疗。
[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.

【学位授予单位】：天津工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08

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