本文選題：生物礦化 + 骨髓基質(zhì)干細(xì)胞�。� 參考：《浙江大學(xué)》2017年博士論文

【摘要】：磷酸鈣作為生物體中骨和牙齒的主要無機(jī)成分,受到化學(xué)、材料和生物醫(yī)學(xué)等多個(gè)學(xué)科的廣泛關(guān)注。基于納米磷酸鈣良好的生物相容性和可降解性,廣泛應(yīng)用于藥物傳送、生物成像和生物醫(yī)學(xué)工程等領(lǐng)域。仿生合成類骨的納米磷酸鈣可以更好的理解生物礦化和生物體硬組織,理解骨分級結(jié)構(gòu)中片狀羥基磷灰石(HAP)的生物學(xué)功能,而且其特殊的性能還可能提高疾病治療的效果和質(zhì)量。然而至今還沒有合成出具有(100)暴露晶面的片狀HAP。因此,本論文主要關(guān)注類骨納米HAP的合成及探討其在生物礦化中的作用,并研究了其在生物醫(yī)學(xué)和骨組織工程中的應(yīng)用。本論文分五章,主要內(nèi)容如下：第一章,我們介紹了生物礦化-細(xì)胞-細(xì)胞外基質(zhì)的基本概念。然后綜述了生物體中的納米磷酸鈣、納米磷酸鈣的細(xì)胞生物效應(yīng)及合成。由于納米磷酸鈣具有好的生物安全性及可降解的性能,我們簡述納米磷酸鈣在生物醫(yī)學(xué)中的應(yīng)用。基于上述內(nèi)容,引出本論文的研究目的及意義。第二章,我們了解到生物體骨組織中的HAP納米晶體是超薄的片狀結(jié)構(gòu),并且在與膠原平行的方向上暴露(100)晶面。我們用乙二醇作溶劑合成了一種新型的HAP顆粒(p-HAP),這種顆粒厚度只有2-4 nm,這個(gè)厚度與骨中的HAP的厚度相似。另外, p-HAP的高分辨透射結(jié)果顯示p-HAP的暴露晶面為(100)晶面,意味著p-HAP的暴露晶面與骨中HAP的暴露晶面相同。p-HAP具有與骨組織HAP相同的晶體形貌和晶面取向,是類骨HAP。同時(shí),我們還合成了另外兩種棒狀HAP(t-HAP和n-HAP),三種HAP的骨髓基質(zhì)干細(xì)胞(MSCs)毒性測試結(jié)果說明三種顆粒均具有很好的生物安全性。第三章,我們采用第二章介紹的類骨p-HAP和另外兩種棒狀HAP(t-HAP和n-HAP)來制備不同的HAP膜作為體外細(xì)胞實(shí)驗(yàn)的基底。這三種膜用來評價(jià)超薄礦物對細(xì)胞生物活性和骨誘導(dǎo)能力的影響。我們發(fā)現(xiàn)類骨p-HAP膜比t-HAP膜和n-HAP膜更促進(jìn)MSCs細(xì)胞的貼壁和增殖。同時(shí),堿性磷酸酶(ALP)活性和定量聚合酶鏈反應(yīng)(PCR)的結(jié)果也發(fā)現(xiàn)類骨p-HAP膜對MSCs細(xì)胞表現(xiàn)出最佳的骨誘導(dǎo)能力。具有(100)暴露晶面的p-HAP膜比(001)為主要暴露晶面的棒狀HAP膜更好的促進(jìn)細(xì)胞的生物活性和成骨分化,意味著MSCs能夠識別HAP的(100)晶面。研究發(fā)現(xiàn)這種晶面選擇性依賴于纖粘連蛋白(FN)在(100)晶面上的選擇性吸附。FN蛋白是對細(xì)胞貼壁起重要作用的功能性蛋白。分子動(dòng)力學(xué)模擬證實(shí)了FN在(100)晶面上的吸附優(yōu)于(001)。我們的實(shí)驗(yàn)結(jié)果說明自然界骨組織選擇具有(100)暴露晶面的片狀HAP作為其基本結(jié)構(gòu)單元是明智的選擇,這對于在骨形成過程中MSCs的成骨分化有明顯的促進(jìn)作用。第四章,我們把類骨片狀HAP與聚乳酸(PLA)結(jié)合形成p-HAP/PLA復(fù)合支架。合成純PLA、t-HAP/PLA和n-HAP/PLA支架作為對照組。從體內(nèi)和體外兩個(gè)方面來考察支架的生物相容性和骨誘導(dǎo)能力。研究檢測了支架對體外人源骨髓基質(zhì)干細(xì)胞(hMSCs)增殖和成骨分化的影響。結(jié)果顯示p-HAP/PLA能夠?qū)MSCs的增殖及成骨分化起到促進(jìn)作用。而且,與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誘導(dǎo)性。隨后,我們將支架材料分別植入SD大鼠顱骨骨缺損的實(shí)驗(yàn)動(dòng)物體內(nèi)來評價(jià)p-HAP/PLA的骨再生能力,不做任何處理的骨缺損動(dòng)物作為空白對照。在術(shù)后8和16周,用micro CT檢測和組織學(xué)分析方法評價(jià)支架的骨修復(fù)效果。所有的實(shí)驗(yàn)結(jié)果均顯示p-HAP/PLA復(fù)合支架比其他支架能夠更好的促進(jìn)骨缺損部位的修復(fù)。此結(jié)果預(yù)示類骨p-HAP的加入能夠提高支架的骨修復(fù)速度和質(zhì)量,p-HAP/PLA在材料誘導(dǎo)的骨修復(fù)和骨組織工程中具有應(yīng)用潛能。第五章,我們總結(jié)本論文的研究。合成了類骨p-HAP,并研究了類骨p-HAP的生物學(xué)功能,進(jìn)而將類骨p-HAP引入PLA形成復(fù)合支架在體內(nèi)和體外檢測p-HAP/PLA的骨修復(fù)情況。最后,對本論文的工作不足進(jìn)行分析并指出今后研究中需要解決的問題。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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