动态液体—静电纺新型三维支架的制备、表征及生物相容性

发布时间：2018-03-25 03:35

本文选题：动态液体-静电纺　切入点：三维支架　出处：《东华大学》2012年硕士论文

【摘要】：骨是人体重要的支撑结构,具有保护器官和执行造血等功能。大块骨缺损是骨科面临的一大难题。组织工程为骨缺损的修复提出了新途径。支架、种子细胞、生长因子是组织工程的三要素。骨组织工程支架基本目的是尽可能模仿和接近松质骨的机械性能和结构。骨细胞外基质(extracellular matrix, ECM)由非矿化的有机组分和矿化的刚性无机物质组成。由于ECM在细胞的生长过程中起着至关重要的调控作用,构建具有类似ECM结构与功能的支架是组织工程的关键技术之一。本论文采用动态液体-静电纺技术制备了两类新型可降解的纳米纤维三维支架,分别采用磷酸-钙液交替浸渍法和模拟体液(SBF)浸渍法仿生矿化,模拟天然骨组织的结构和成分。使用MC3T3-E1细胞研究两类支架的生物相容性,为骨组织工作支架的构建提供新的方法。主要研究内容如下： (1)首次采用改良的动态液体-静电纺技术制备了丝素蛋白(silk fibroin,SF)与P(LLA-CL)混纺纳米纱和单纯P(LLA-CL)的纳米纱三维支架,用磷酸-钙液交替浸渍法仿生矿化。通过扫描电子显微镜和红外光谱、X射线衍射光谱分析矿化前后两种纳米纱的结构和理化性质。研究结果表明,纳米纱是由多根纳米纤维成股而形成的。在组成单元上,P(LLA-CL)纳米纱比SF与P(LLA-CL)混纺的纳米纱直径更大,同时宏观结构更规则。SF与P(LLA-CL)的混纺后,通过水的接收,能够使得SF快速转变为不溶于水的B-折叠的silk II结晶结构。红外光谱、X射线衍射光谱分析证实通过磷酸-钙液交替浸渍法能够使得支架上沉积羟基磷灰石颗粒。细胞相容性实验表明,由纳米纤维形成的纳米纱极大地影响细胞的粘附和迁移行为。纯聚合物材料的P(LLA-CL)由于疏水的表面缺乏细胞粘附的功能性基团,不利于细胞增殖,混合有SF的纳米纱支架能够有效促进细胞增殖。MC3T3-E1细胞在矿化前后两种支架上都能够增殖,其中P(LLA-CL)/SF、P(LLA-CL)-HA和P(LLA-CL)/SF-HA支架上的细胞数量出现显著性增殖(P0.05)。通过HE染色证明,在矿化前后的纳米纱支架上都发现了细胞的长入,细胞沿着纤维生长,呈现沿着纤维的走向迁移,遍布了整个支架中。本实验结果证明,改良的动态液体-静电纺技术能够提供一种用于组织工程三维支架材料的制备方法,制备的三维纳米纱能够克服传统静电纺制备出的纳米纤维膜导致细胞难以长入支架内部的缺点,有望应用于骨组织修复。 (2)为了增强三维结构的纳米纤维的力学性能,本研究利用纤维增强的原理结合冷冻干燥技术,制备了新型P(LLA-CL)和P(LLA-CL)/SF纳米纱增强丝素蛋白的复合型三维支架,使用75%乙醇蒸汽诱导支架的水不溶性。采用10×SBF浸渍法来进行支架的仿生矿化。结果表明纤维增强这一方式对复合支架的孔隙率,力学性能有着巨大的影响。随着纳米纱的加入,相比纯SF支架,两种纤维增强型复合支架的孔隙率有一定程度的下降,同时力学性能得到明显的增加。纯SF支架的弹性模量为1.2848±0.1107MPa,孔隙率约为86.28%。PLCL-R-SF支架的弹性模量增加到1.17017±O.1619MPa,孔隙率降低至84.97%,而PLCL/SF-R-SF支架的弹性模量的弹性模量增加到1.7265±0.5042MPa,孔隙率降低至82.82%。矿化沉积的结晶颗粒的钙磷比(Ca/P)为1.56,接近羟基磷灰石的Ca/P=1.67。红外光谱、X射线衍射光谱分析进一步证实通过10×SBF浸渍法能够使得支架上沉积球型的羟基磷灰石颗粒。PLCL-R-SF-HA和PLCL/SF-R-SF-HA支架上的细胞数量有了显著性的增加(P0.05),同时MC3T3-E1在支架上已经分泌出了自身的明显的细胞外基质。说明在纳米水平上球状的羟基磷灰石颗粒可能会减少MC3T3-E1细胞在支架上早期的增殖,但是在后期羟基磷灰石对细胞增殖起了明显的促进作用。本实验结果证明,纤维增强这一方式能够极大的提高三维纳米纤维支架的力学强度,纤维增强型复合支架在骨组织修复中具有良好的应用潜能。
[Abstract]:Bone is an important supporting structure of human body, can protect organs and hematopoietic function. The execution of large bone defects is a big problem in the Department of orthopedics face. Tissue engineering for the repair of bone defects and puts forward a new way. Scaffolds, seed cells, growth factors are the three elements of tissue engineering. The basic purpose of bone tissue engineering scaffold mechanical properties biomimic the structure and cancellous bone. The bone extracellular matrix (extracellular, matrix, ECM) by non mineralized rigid inorganic substances and mineralization of organic fractions. Because ECM play a vital role in the process of cell growth, construction of stents with similar structure and function of ECM is one of the key technology of tissue engineering.
In this paper, two kinds of novel biodegradable nanofiber scaffolds prepared by liquid electrospinning technique, respectively using phosphoric acid and calcium liquid alternate immersion method and simulated body fluid (SBF) immersion method of biomimetic mineralization, structure and composition of the simulated natural bone tissue compatibility. The use of MC3T3-E1 cell research two kinds of biological scaffold and provide a new method for constructing bone tissue scaffold work. The main contents are as follows:
(1) for the first time in the preparation of silk fibroin by dynamic modified liquid electrospinning technology (silk fibroin SF) and P (LLA-CL) blended yarn and pure nano P (LLA-CL) nano yarn scaffold, phosphate - calcium liquid alternate immersion biomimetic mineralization by scanning electron microscopy and infrared spectroscopy. X ray diffraction spectrum analysis of mineralization structure before and after the two nanometer yarn and physicochemical properties. The results show that the nano yarn is formed by a plurality of nano fiber strands. In the unit, P (LLA-CL) than SF and P nano yarn (LLA-CL) nano blended yarn diameter is larger, while the macro structure more rules.SF and P (LLA-CL) of the blend, through the water receiver, can make SF silk II a rapid change of crystal structure of folding as insoluble B-. Infrared spectroscopy, X ray diffraction spectrum analysis confirmed by calcium phosphate liquid alternate immersion method can make the deposition of hydroxyapatite scaffold on Particles. The cell compatibility experiment showed that nano fiber yarn formed by nanoparticles greatly affects cell adhesion and migration behavior of pure polymer materials. P (LLA-CL) due to the lack of hydrophobic surface functional groups is not conducive to cell adhesion, cell proliferation, mixed with nano SF yarn stent can effectively promote the cell proliferation of.MC3T3-E1 cells can the proliferation in mineralization before and after the two support, including P (LLA-CL) /SF, P (LLA-CL) -HA and P (LLA-CL) the number of cells on /SF-HA scaffolds induced significant proliferation (P0.05). Proved by HE staining in the nano yarn before and after stent mineralization were found on the growth of cells, cell along the fiber growth, showing migration along the fiber direction throughout the scaffold. The experimental results show that the dynamic modified liquid electrospinning technology can provide a method for preparing three-dimensional scaffold for tissue engineering, system The three dimensional nano yarn prepared by this technology can overcome the shortcomings of the traditional nanofiber membrane produced by electrospinning and cause cells to not grow into the inner part of the scaffold, which is expected to be applied to bone tissue repair.
(2) in order to enhance the mechanical properties of nano fiber three-dimensional structure, this study utilizes the principle of fiber reinforced with freeze drying technology to prepare P model (LLA-CL) and P (LLA-CL) /SF nano yarn reinforced composite scaffold of silk protein scaffolds, induced by using 75% ethanol steam water insoluble. To support the biomimetic mineralization with 10 x SBF impregnation method. The results show that the porosity of the fiber reinforced composite scaffolds, have great effects on mechanical properties. With the addition of nano yarn, compared with pure SF stent, two kinds of fiber reinforced composite scaffold porosity decreased to a certain extent, and mechanical properties are increased obviously. The elastic modulus of pure SF scaffold was 1.2848 + 0.1107MPa, the porosity is about 86.28%.PLCL-R-SF elastic modulus of the scaffolds increased to 1.17017 + O.1619MPa, the porosity decreased to 84.97%, while the elastic modulus of PLCL/SF-R-SF support The elastic modulus increased to 1.7265 + 0.5042MPa, the porosity crystalline particles to 82.82%. mineralization of calcium and phosphorus ratio (Ca/P) for 1.56 Ca/P=1.67., close to the infrared spectrum of hydroxyapatite, X ray diffraction spectrum analysis further confirmed by 10 * SBF impregnation method can make the number of.PLCL-R-SF-HA PLCL/SF-R-SF-HA stent and hydroxyapatite particles deposited on the ball type the cells are significantly increased (P0.05), and MC3T3-E1 in the scaffold has produced a distinct extracellular matrix. Its hydroxyl apatite particles in spherical nanometer level may reduce the proliferation of MC3T3-E1 cells in the early support, but in the late stage of hydroxyapatite on cell proliferation was significantly promoted. The experimental result shows that the fiber reinforced this way can greatly improve the three-dimensional nanofiber scaffold mechanical strength, fiber The reinforced composite scaffold has a good potential in the repair of bone tissue.

【学位授予单位】：东华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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