模拟体液生物矿化产物与成骨细胞行为的关联研究

发布时间：2018-01-05 12:03

本文关键词：模拟体液生物矿化产物与成骨细胞行为的关联研究　出处：《北京化工大学》2015年硕士论文　论文类型：学位论文

【摘要】：骨缺损的发生给患者带来了极大的不便,自体骨移植容易造成二次损伤,异体骨移植带来免疫排斥反应。随着组织工程的发展,骨组织工程使得人工骨的构建能够解决上述不足。在选用生物相容性较高的材料的基础上,通过静电纺丝手段制备接近细胞外基质的结构能够赋予支架更高的生物相容性。为了提高人工骨的骨诱导性能,研究者把羟基磷灰石(HA),磷酸三钙(β-TCP)粒子等无机陶瓷粒子引入到支架中,并目.取得了预期的效果。干粉状态的纳米无机陶瓷粒子很难均匀分散到用于静电纺丝的聚合物溶液中,即使是经过了超声分散和表面改性,纳米粒子的团聚现象依然存在。生物矿化最初是作为评价材料的成骨性能的手段,成骨性能好的材料在模拟体液中能够形成均匀钙磷盐层,分散效果优于将无机陶瓷粒子直接分散到纺丝液中的方法。研究者尝试使用不同种类的模拟体液,不同矿化条件下在聚合物材料表面引入钙磷盐,最终得到了具有促进成骨分化的有机-无机的支架。但是,研究者们采用的矿化基体的组成、结构不同,矿化用的模拟体液种类不同,矿化时间及其他矿化因素也存在差异,因此得到的钙磷盐虽然同属于钙磷盐,但在微观组成、结构上存在着差异,在和细胞培养基作用过程中,这些差异会导致钙磷盐的组成、结构、溶出的离子对细胞成骨分化产生影响。这就导致了研究者们通过矿化手段得到的钙磷盐无法进行系统比较,无法得出矿化参数的改变对钙磷盐形成的影响,最终对成骨分化的影响；其次研究者侧重于矿化产物的获得,很少关注矿化各个阶段的产物特点对细胞的影响。我们选择生物相容性良好的聚左旋聚乳酸/明胶作为矿化的聚合物基体,通过静电纺丝于段制备的具有ECM结构的支架材料。矿化体系选用矿化时间相对较短、矿化产物变化明显的改性的i-5SBF随着矿化的进行,每6小时从矿化体系中取得带有钙磷盐的支架材料。为了考察不同矿化阶段的产物在细胞培养体系中的转变过程,排除细胞培养基中蛋白质和其他有机物对材料的影响,我们选择HANKS缓冲溶液作为材料的溶出体系,增加去离子水的溶出体系作为参照组,模仿细胞培养中的换液操作,在第1,3,6,9,12,15,18大更换新的溶出液。在换液的时间点,从溶出体系中取出材料,通过SEM、XRD、FT-IR、称重观察材料表面钙磷盐组成、结构的变化,并且通过ICP测量更换出的溶出液中Ca2+浓度的变化,系统的表征矿化到不同阶段产物对溶出体系的影响,找出对细胞影响可能的因素。再将矿化到不同阶段的材料和成骨细胞共培养,考察不同材料对成骨细胞增殖、ALP活性、Ⅰ型胶原合成的影响,在成骨细胞增殖和ALP活性实验中增设Transwell中材料和成骨细胞共培养的方式考察培养体系中钙离子浓度对成骨细胞的影响。结合溶出实验和细胞培养实验,我们不仅可以得到矿化到不同阶段的产物对细胞产生怎样的影响,通过控制矿化参数可以影响材料的成骨分化性能,也能够为其他矿化体系提供评价模型和参考。
[Abstract]:The occurrence of bone defect has brought great inconvenience to patients, autologous bone graft is easy to cause two damage, caused allograft immune rejection. With the development of tissue engineering, bone tissue engineering makes the construction of artificial bone can solve the above problems. Based on the selection of biocompatible material with higher structure, through electrostatic spinning the preparation approach of extracellular matrix can give higher compatibility support. In order to improve the biological artificial bone bone induction properties, the researchers put hydroxyapatite (HA), calcium phosphate (beta -TCP) particles of inorganic ceramic particles into the bracket, and the head. The expected results were achieved. The nano inorganic the dry state hard ceramic particles uniformly dispersed into polymer solution for electrospinning, even after ultrasonic dispersion and surface modification, agglomeration of nanoparticles still exist. Biological mineralization At first as a means to evaluate the performance of bone material, bone material with good properties can form a uniform layer of calcium and phosphorus in simulated body fluid, the dispersion effect is better than that of inorganic ceramic particles directly dispersed into the method of spinning liquid. SBF try using different types of research, different mineralization conditions in the calcium phosphate salt is introduced the surface of polymer materials, obtained with the promotion of the supports of organic inorganic bone differentiation. However, the researchers used the mineralized matrix of the structure is different, with different types of simulated body fluid mineralization, there are differences in mineralization time and other mineralization factors, so the calcium phosphate belong to calcium phosphate salt but, in the microcosmic composition, structural differences, and in the cell culture process, these differences will lead to calcium phosphate salt composition, structure, ion dissolution affect cell osteogenic differentiation Ring. This leads to the comparison of the researchers obtained by means of the mineralization of calcium phosphate can influence system, unable to obtain the parameters on the mineralization of calcium phosphate salt formation, the ultimate impact on the osteogenic differentiation; secondly get researchers focused on mineralization products, very little attention to the product characteristics of each stage of mine on the cell. We choose the good biocompatibility of poly PLLA / gelatin as polymer matrix mineralization, scaffold material with ECM structure by electrospinning in preparation. The mineralization system mineralization in a relatively short time, mineral products change obvious modification of i-5SBF with mineralization, every 6 the system has hours from mineralization of calcium phosphate scaffolds with different mineralization stages. In order to investigate the product in the transformation process in the system of exclusion of cultured cells, cell culture medium and other protein Effect of organic materials, we choose the HANKS buffer solution as the stripping system of material, increase the deionized water dissolution system as reference and imitation in cell culture medium change operation, in the replacement of the 1,3,6,9,12,15,18 solution. The change of liquid time, remove the material from the stripping system by SEM, XRD, FT-IR, weighing to observe the surface of calcium phosphate salt composition, structural changes, and measured by the ICP to replace a dissolution of the concentration of Ca2+ in the characterization of mineralization system to products in different stages of dissolution system, find out the factors of cell effect. Then to mineralization the different stages of the materials are co cultured with osteoblasts, the effects of different materials on the proliferation of bone cells, the activity of ALP, effects of type I collagen synthesis, in addition of Transwell material and osteoblasts were cultured in the bone cell proliferation and ALP activity in the experiment Type inspection and training effects of calcium ion concentration in the system of osteoblasts. Combined with dissolution experiments and cell culture experiments, we can not only get to the products of different stages of mineralization and what impact on the cell, through the control of mineralization parameters can affect the osteogenic differentiation of materials, and can also provide reference for other evaluation model mineralization system.

【学位授予单位】：北京化工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R318.08

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