多形貌纳米羟基磷灰石的制备及其细胞相容性研究

发布时间：2018-04-28 21:11

本文选题：羟基磷灰石 + 有机分子　；参考：《暨南大学》2012年硕士论文

【摘要】：在生物材料研究领域中，羟基磷灰石（HAP）由于其化学成分结构与骨牙中的无机成分高度相似，在硬组织替换与修复领域中得到广泛的应用。生物矿化形成的自然骨和牙中HAP的形貌分别为纳米片状和六方棒状，与常规人工合成HAP形态差异较大。本研究基于生物矿化的基本原理，采用水热反应法，以富含氨基和羧基的有机小分子甘氨酸，乙二胺，谷氨酸，柠檬酸钠为模型分子，研究小分子基团对HAP的晶体生长形貌的影响；探讨不同形貌HAP的制备方法和形成机理。采用透射电子显微镜（TEM）、X-射线衍射仪（XRD）、拉曼（Laman）和傅立叶变换红外光谱仪（FTIR）分析了合成产物的形貌、结构和组成。实验结果表明：乙二胺的诱导下合成了宽30～50nm，长宽比3～5的片状HAP晶体；甘氨酸诱导合成的HAP呈六方棒状，长500～1500nm，直径50～100nm；谷氨酸诱导合成的HAP由纳米棒状组装成的片状结构，长500～2000nm，宽50～200nm。而柠檬酸钠诱导合成的HAP也呈片状，长200～500nm，宽20～50nm，尺寸均匀。对照空白实验可知，小分子上的极性基团通过静电吸附或分子占位显著影响HAP的形貌。带正电的氨基可能占据Ca2+的空缺位置，特别是在（001）面，故乙二胺调控下，HAP在(001)面的生长受到抑制，形成片状。带负电荷的羧基与Ca2+强烈的配位作用及分子间的相互作用，导致甘氨酸、谷氨酸和柠檬酸分子形成了不同的形貌HAP。在谷氨酸分子调控下，纳米棒HAP（100）面的Ca2+吸附羧基，谷氨酸在（100）面形成C—N—C分子层，通过羧基与Ca2+的作用组装成纳米有序排列的片状HAP晶体。含有3个羧基的柠檬酸与Ca2+的强配位作用，防止了HAP的厚化，，使其保持片状形貌。该研究从一定角度证实了自然骨形成过程中柠檬酸干扰骨质磷灰石的形成稳定其片状结构可能存在的调控机制。本研究对上述不同形貌的纳米HAP材料的生物相容性进行了研究。通过颗粒悬液检测材料的细胞毒性，结果表明所合成的几种不同形貌的纳米HAP材料随着颗粒浓度的提高，细胞的活性降低。当颗粒浓度达到10μg/ml以下，没有表现出明显的细胞毒性。在检测浓度范围内（50～5μg/ml），纳米晶粒形貌的不同未导致细胞活性的差异。研究了纳米HAP对成骨细胞的增殖、ALP的表达和细胞形态的影响。从MTT统计结果表明，随着纳米HAP浓度的增加，成骨细胞活性逐渐下降。纳米颗粒尺寸增大，成骨细胞活性下降。纳米HAP抑制了成骨细胞的生长，促进成骨细胞ALP的表达。SEM和激光共聚焦显微镜的结果表明不同样品对细胞形态的影响并没有明显的差异。
[Abstract]:Hydroxyapatite (HAP) has been widely used in the field of hard tissue replacement and repair because of its high similarity in chemical composition and inorganic composition in bone teeth in the field of biomaterial research. The morphology of HAP in the natural bone and teeth formed by biomineralization was nanoscale and hexagonal, which was different from that of conventional synthetic HAP. Based on the principle of biomineralization, the model molecules of glycine, ethylenediamine, glutamic acid and sodium citrate, which are rich in amino and carboxyl groups, were used in this study. The effects of small molecular groups on the growth morphology of HAP were studied, and the preparation methods and formation mechanism of HAP with different morphologies were discussed. The morphology, structure and composition of the synthesized products were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). The experimental results showed that HAP crystals with width of 30 ~ 50 nm and aspect ratio of 3 ~ 5 were synthesized under the induction of ethylenediamine, HAP synthesized by glycine was hexagonal, 500 ~ 1 500 nm in length and 50 ~ 100 nm in diameter, and HAP synthesized by glutamate was composed of nanorods of 50 000 nm in length and 50 ~ 200 nm in width. The HAP induced by sodium citrate was also flake, with a length of 200 ~ 500 nm and a width of 20 ~ 50 nm with a uniform size. Compared with the blank experiments, the polar groups on the small molecules have significant influence on the morphology of HAP by electrostatic adsorption or molecular occupation. The positively charged amino group may occupy the vacant position of Ca2, especially on the surface of Ca2. Therefore, the growth of Ca2 on the surface of Ca2 is inhibited under the control of ethylenediamine. The strong coordination and intermolecular interaction between negatively charged carboxyl groups and Ca2 lead to the formation of different morphologies of glycine, glutamate and citric acid molecules. Under the control of glutamate molecule, Ca2 adsorbs carboxyl group on the surface of nanorod HAP-100, and glutamic acid forms C-N-C molecular layer on the surface of HAP-100). Through the interaction of carboxyl group and Ca2, the ordered sheet HAP crystal is assembled. The strong coordination of citric acid containing three carboxyl groups with Ca2 prevents the thickening of HAP and keeps it flake. This study confirmed the possible regulatory mechanism of citric acid interfering with the formation of bone apatite in the process of natural bone formation in order to stabilize its lamellar structure. The biocompatibility of nanometer HAP materials with different morphologies was studied in this paper. The cytotoxicity of the material was detected by particle suspension. The results showed that the cell activity of several nano-sized HAP materials with different morphologies decreased with the increase of particle concentration. When the particle concentration was below 10 渭 g/ml, there was no obvious cytotoxicity. In the concentration range of 50 渭 g / ml, the difference of the morphology of nanocrystalline did not lead to the difference of cell activity. The effects of nanometer HAP on the expression and morphology of osteoblasts were studied. The results of MTT showed that the activity of osteoblasts decreased with the increase of HAP concentration. The size of nanoparticles increased and the activity of osteoblasts decreased. Nanometer HAP inhibited the growth of osteoblasts and promoted the expression of ALP in osteoblasts. The results of laser confocal microscopy showed that there was no significant difference in the effect of different samples on the morphology of osteoblasts.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08;TB383.1

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