类骨羟基磷灰石的合成与应用

发布时间：2018-05-27 17:43

本文选题：生物矿化 + 骨髓基质干细胞　；参考：《浙江大学》2017年博士论文

【摘要】：磷酸钙作为生物体中骨和牙齿的主要无机成分,受到化学、材料和生物医学等多个学科的广泛关注。基于纳米磷酸钙良好的生物相容性和可降解性,广泛应用于药物传送、生物成像和生物医学工程等领域。仿生合成类骨的纳米磷酸钙可以更好的理解生物矿化和生物体硬组织,理解骨分级结构中片状羟基磷灰石(HAP)的生物学功能,而且其特殊的性能还可能提高疾病治疗的效果和质量。然而至今还没有合成出具有(100)暴露晶面的片状HAP。因此,本论文主要关注类骨纳米HAP的合成及探讨其在生物矿化中的作用,并研究了其在生物医学和骨组织工程中的应用。本论文分五章,主要内容如下：第一章,我们介绍了生物矿化-细胞-细胞外基质的基本概念。然后综述了生物体中的纳米磷酸钙、纳米磷酸钙的细胞生物效应及合成。由于纳米磷酸钙具有好的生物安全性及可降解的性能,我们简述纳米磷酸钙在生物医学中的应用。基于上述内容,引出本论文的研究目的及意义。第二章,我们了解到生物体骨组织中的HAP纳米晶体是超薄的片状结构,并且在与胶原平行的方向上暴露(100)晶面。我们用乙二醇作溶剂合成了一种新型的HAP颗粒(p-HAP),这种颗粒厚度只有2-4 nm,这个厚度与骨中的HAP的厚度相似。另外, p-HAP的高分辨透射结果显示p-HAP的暴露晶面为(100)晶面,意味着p-HAP的暴露晶面与骨中HAP的暴露晶面相同。p-HAP具有与骨组织HAP相同的晶体形貌和晶面取向,是类骨HAP。同时,我们还合成了另外两种棒状HAP(t-HAP和n-HAP),三种HAP的骨髓基质干细胞(MSCs)毒性测试结果说明三种颗粒均具有很好的生物安全性。第三章,我们采用第二章介绍的类骨p-HAP和另外两种棒状HAP(t-HAP和n-HAP)来制备不同的HAP膜作为体外细胞实验的基底。这三种膜用来评价超薄矿物对细胞生物活性和骨诱导能力的影响。我们发现类骨p-HAP膜比t-HAP膜和n-HAP膜更促进MSCs细胞的贴壁和增殖。同时,碱性磷酸酶(ALP)活性和定量聚合酶链反应(PCR)的结果也发现类骨p-HAP膜对MSCs细胞表现出最佳的骨诱导能力。具有(100)暴露晶面的p-HAP膜比(001)为主要暴露晶面的棒状HAP膜更好的促进细胞的生物活性和成骨分化,意味着MSCs能够识别HAP的(100)晶面。研究发现这种晶面选择性依赖于纤粘连蛋白(FN)在(100)晶面上的选择性吸附。FN蛋白是对细胞贴壁起重要作用的功能性蛋白。分子动力学模拟证实了FN在(100)晶面上的吸附优于(001)。我们的实验结果说明自然界骨组织选择具有(100)暴露晶面的片状HAP作为其基本结构单元是明智的选择,这对于在骨形成过程中MSCs的成骨分化有明显的促进作用。第四章,我们把类骨片状HAP与聚乳酸(PLA)结合形成p-HAP/PLA复合支架。合成纯PLA、t-HAP/PLA和n-HAP/PLA支架作为对照组。从体内和体外两个方面来考察支架的生物相容性和骨诱导能力。研究检测了支架对体外人源骨髓基质干细胞(hMSCs)增殖和成骨分化的影响。结果显示p-HAP/PLA能够对hMSCs的增殖及成骨分化起到促进作用。而且,与PLA、t-HAP/PLA和n-HAP/PLA支架相比,p-HAP/PLA具有最好的生物相容性和骨诱导性。随后,我们将支架材料分别植入SD大鼠颅骨骨缺损的实验动物体内来评价p-HAP/PLA的骨再生能力,不做任何处理的骨缺损动物作为空白对照。在术后8和16周,用micro CT检测和组织学分析方法评价支架的骨修复效果。所有的实验结果均显示p-HAP/PLA复合支架比其他支架能够更好的促进骨缺损部位的修复。此结果预示类骨p-HAP的加入能够提高支架的骨修复速度和质量,p-HAP/PLA在材料诱导的骨修复和骨组织工程中具有应用潜能。第五章,我们总结本论文的研究。合成了类骨p-HAP,并研究了类骨p-HAP的生物学功能,进而将类骨p-HAP引入PLA形成复合支架在体内和体外检测p-HAP/PLA的骨修复情况。最后,对本论文的工作不足进行分析并指出今后研究中需要解决的问题。
[Abstract]:Calcium phosphate is the main inorganic component of bone and teeth in organism. It is widely concerned in many disciplines, such as chemistry, material and biomedicine. Based on the good biocompatibility and degradability of calcium phosphate, it is widely used in the fields of drug delivery, bioimaging and biomedical engineering. Bionic synthesis of calcium phosphate nanoscale nanoparticles can be used. In order to better understand biomineralization and biological hard tissue, understand the biological function of HAP in bone grading structure, and its special performance may also improve the effect and quality of disease treatment. However, there has not been a (100) Bao Lujing flake HAP. so far, this paper mainly focuses on bone like nano HA The synthesis and study of P's role in biomineralization and its application in biomedical and bone tissue engineering are studied. This thesis is divided into five chapters. The main contents are as follows: in Chapter 1, we introduced the basic concepts of biomineralization - cell - extracellular matrix. Then, the nano calcium phosphate and calcium phosphate nanoscale cells in the biological body were summarized. Biological effects and synthesis. Because of the good biological safety and biodegradability of nano calcium phosphate, we briefly describe the application of nano calcium phosphate in biomedicine. Based on the above content, the purpose and significance of this research are introduced. In the second chapter, we understand that the HAP nanocrystals in the bone tissue of the organism are thin sheet structures, and And in the direction of parallel to collagen (100), we have synthesized a new type of HAP particle (p-HAP) with ethylene glycol, which is only 2-4 nm in thickness. This thickness is similar to the thickness of HAP in bone. In addition, the high resolution transmission of p-HAP shows that the exposed crystal surface of p-HAP is (100) crystal surface, meaning the exposed crystal of p-HAP. .p-HAP has the same crystal morphology and crystal surface orientation as bone tissue HAP, which is the same as bone tissue HAP. We also synthesize two other kinds of rod like HAP (t-HAP and n-HAP), three HAP's bone marrow stromal cells (MSCs) toxicity test results show that three kinds of particles have good biological safety. Third chapter, I We used second chapters of p-HAP and two other rod like HAP (t-HAP and n-HAP) to prepare different HAP membranes as the basement of cell experiment in vitro. These three membranes are used to evaluate the effect of ultrathin minerals on cell bioactivity and bone induction. We found that the p-HAP membrane of bone like membrane promotes the adherence of MSCs cells more than the t-HAP membrane and n-HAP membrane. At the same time, the results of alkaline phosphatase (ALP) activity and quantitative polymerase chain reaction (PCR) also found that the bone like p-HAP membrane showed the best bone induction ability for MSCs cells. The p-HAP membrane with (100) exposed crystal surface was better to promote cell biological activity and osteogenic differentiation than (001) as the main exposed crystal surface, meaning MSCs energy. Enough to identify the (100) surface of HAP. The study found that the selective adsorption of this crystal surface on fibronectin (FN) on the (100) crystal surface is a functional protein that plays an important role in cell adhesion. Molecular dynamics simulation confirms that the absorption of FN on (100) surface is superior to (001). Our experimental results indicate the bone tissue in nature. The selection of flake HAP with (100) exposing crystal surface as its basic structural unit is a wise choice, which has an obvious promoting effect on osteogenic differentiation of MSCs during bone formation. In Chapter fourth, we combine bone like HAP with polylactic acid (PLA) to form a p-HAP/PLA composite support. Pure PLA, t-HAP/PLA, and n-HAP/PLA scaffolds are used as control Study the effects of scaffolds on the proliferation and osteogenic differentiation of human bone marrow stromal cells (hMSCs) in vitro. The results showed that p-HAP/PLA could promote the proliferation and osteogenic differentiation of hMSCs and promote the proliferation and osteogenic differentiation of hMSCs. Moreover, it was associated with PLA, t-HAP/PLA and n-HAP/PLA branches. P-HAP/PLA had the best biocompatibility and bone inducibility. Then, we implanted the scaffold materials into the experimental animals of the SD rat skull bone defect to evaluate the bone regeneration ability of p-HAP/PLA, without any treated bone defect animals as blank control. 8 and 16 weeks after the operation, micro CT detection and histological analysis were used. The results of all experimental results showed that the p-HAP/PLA composite scaffold could better promote the repair of bone defect than other scaffolds. This result indicates that the addition of p-HAP like bone can improve the speed and quality of the bone repair of the scaffold, and the p-HAP/PLA can be applied in the bone repair and bone tissue engineering of the material. In the fifth chapter, we summarize the study of this thesis. We synthesized the bone like p-HAP, and studied the biological function of the bone like p-HAP. Then the bone like p-HAP was introduced into PLA to form a composite scaffold to detect the bone repair of p-HAP/PLA in vivo and in vitro. Finally, the shortcomings of the work in this paper were analyzed and the problems to be solved in the future were pointed out.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R318.08

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