本文選題：介孔硅　切入點：功能化　出處：《東華大學》2016年博士論文

【摘要】：由外傷、腫瘤、感染和疾病等因素造成的骨缺損嚴重影響患者的身心健康和生活質(zhì)量。傳統(tǒng)的自體骨和異體骨移植仍然是臨床上治療骨缺損的常用方案,但由于這些方法存在自身的局限性限制了它們的廣泛應用,難以滿足龐大的臨床需求,因此開發(fā)具有優(yōu)良生物活性的骨修復材料成為研究熱點。介孔硅納米粒子(mesoporous silica nanoparticles,MSNs)是一種納米級的無機粒子,具有許多獨特的優(yōu)點,如大的比表面積和孔體積、可調(diào)的粒徑和孔徑以及易修飾的表面特性等,在生物醫(yī)學領(lǐng)域具有較好的應用前景。當前,研究者們開始聚焦MSNs在骨組織工程的應用。本課題以MSNs為基體,根據(jù)其表面易改性、可封裝客體分子等特點,構(gòu)建可用于骨缺損修復的納米骨修復材料以及多孔復合支架材料,然后對所制備的材料的理化性能和體內(nèi)外成骨誘導能力進行研究。本文的研究內(nèi)容概括為以下幾個部分:1.以合成的Nε-芐氧羰基-L-賴氨酸-N-羧酸酐(Lys(Z)-NCA)為單體,利用開環(huán)聚合反應在聚乙烯亞胺(PEI)鏈上生成聚-L-賴氨酸(PLL),制備PLL修飾的PEI共聚物(PEI-PLL)。使用了三種不同分子量的PEI(分子量為1800,10000和25000)作為大分子引發(fā)劑,制備了對應的三種不同PEI-PLL共聚物(PEI-PLL-1.8k,PEI-PLL-10k和PEI-PLL-25k)。通過1H NMR、FTIR等表征技術(shù)證實三種PEI-PLL共聚物的成功合成。然后,分析了三種pei-pll共聚物的緩沖能力、質(zhì)粒dna(pdna)結(jié)合能力和對pdna的dnasei保護能力。細胞成活率實驗結(jié)果顯示,三種pei-pll共聚物的細胞毒性都低于分子量為25000的純pei(pei-25k)。通過熒光素酶表達、熒光顯微鏡觀察和流式細胞術(shù)研究pei-pll共聚物的轉(zhuǎn)染效率,結(jié)果顯示pei-pll-25k的轉(zhuǎn)染效率高于其他兩種pei-pll共聚物,同時也比pei-25k表現(xiàn)更高的轉(zhuǎn)染效率。酶聯(lián)免疫吸附測定(elisa)實驗表明,pei-pll-25k能夠有效介導bmp-2基因進入bmscs并表達bmp-2蛋白。而且,堿性磷酸酶(alp)表達和vonkossa染色結(jié)果顯示,pei-pll-25k介導bmp-2基因轉(zhuǎn)染bmscs,能夠增強bmscs的成骨分化。因此,以pei分子量為25000合成的pei-pll-25k共聚物表現(xiàn)更低的細胞毒性和更高的轉(zhuǎn)染效率,可作為非病毒基因載體用于骨相關(guān)疾病的基因治療。2.pei-pll-25k共聚物被證明具有高轉(zhuǎn)染效率和低細胞毒性,通過edc/nhs化學法將其共價接枝在msns表面,并進一步將rgd多肽接枝在粒子表面,制得pei-pll-25k共聚物和rgd修飾的功能化msns(msns-ppr)。然后,制備的msns-ppr能夠通過孔道負載地塞米松(dex)并且可以通過表面異種電荷的靜電作用吸附pdna。細胞相容性和血液相容性結(jié)果顯示,制備的msns-ppr表現(xiàn)出良好的生物相容性,這為msns-ppr作為納米載體奠定了基礎(chǔ)。另外,細胞實驗表明,dex@msns-ppr納米粒子能快速、有效被細胞攝取并分布在細胞核周圍。elisa實驗證實,dex@msns-ppr能夠介導bmp-2基因進入bmscs并成功表達bmp-2蛋白。此外,由alp活性、成骨相關(guān)基因表達和茜素紅染色的體外成骨分化實驗顯示,利用msns-ppr共傳遞bmp-2基因和dex進入bmscs相比單一bmp-2基因傳遞和單一dex傳遞表現(xiàn)更加明顯的成骨誘導作用。因此,同時負載bmp-2基因和dex的msns-ppr表現(xiàn)增強的成骨誘導能力,可作為納米成骨釋放系統(tǒng)應用于骨組織工程。3.通過3-氨基丙基-三乙氧基硅烷(aptes)的連接作用,利用edc/nhs化學法將來源于bmp-2蛋白73-92的一段氨基酸序列接枝在msns表面,得到bmp-2多肽功能化msns(msns-pep)。接著,將dex負載在msns的孔道內(nèi),制備復合bmp-2多肽和dex的納米成骨釋放系統(tǒng)(dex@msns-pep)。體外細胞成活率和細胞攝取實驗表明,bmp-2多肽功能化的msns能提高納米粒子的細胞相容性并增強細胞對納米粒子的攝取。另外,體外誘導實驗,包括alp活性測定、鈣沉積分析和免疫組化染色,結(jié)果證明制備的msns-pep能夠促進bmscs的成骨分化,而且負載dex后,進一步增強對bmscs成骨分化的誘導作用。體內(nèi)異位成骨實驗結(jié)果顯示,在植入后的3周,msns-pep和dex@msns-pep都能誘導新骨的形成。在bmp-2多肽和dex的協(xié)同作用下,dex@msns-pep比msns-pep誘導更加明顯的新骨形成。因此,復合bmp-2多肽和dex的dex@msns-pep納米成骨釋放系統(tǒng)能夠在體外誘導bmscs的成骨分化以及在體內(nèi)誘導異位成骨,在骨組織工程中具有潛在的應用前景。4.為了模擬天然骨組織的有機/無機組成成分,利用msns和明膠制備功能性有機/無機骨修復支架。首先,用MSNs來負載抗生素鹽酸萬古霉素(Van),制得Van@MSNs。然后,在明膠支架的制備過程中加入Van@MSNs納米粒,制備出復合Van@MSNs的明膠復合支架(Van@MSNs/Gelatin)。掃描電鏡(SEM)圖片顯示,隨著MSNs的加入量增多(5%-20%),制得的復合支架的孔徑就越大。同時,MSNs的加入能明顯提高復合支架的壓縮強度,但是當MSNs的加入量從5%提高到20%,其壓縮強度反而逐漸下降,但仍比純Gelatin支架好。定性和定量的抑菌實驗顯示,制備的Van@MSNs/Gelatin復合支架表現(xiàn)良好的抑菌效果。另外,體外細胞實驗表明Van@MSNs/Gelatin復合支架能夠很好地支持BMSCs的粘附、增殖和分化。體內(nèi)感染性骨缺損修復實驗表明,在Van@MSNs/Gelatin復合支架植入12周后,缺損部位得到良好的修復。因此,制備的Van@MSNs/Gelatin復合支架可有效作為感染性骨缺損的修復材料。綜上,本論文主要以MSNs為載體構(gòu)建了復合雙因子的納米成骨釋放系統(tǒng),并進一步將載藥MSNs延伸到復合支架,探究了這些材料在骨修復中的應用。本文的研究工作豐富了MSNs在骨組織工程領(lǐng)域的應用,并在骨缺損的治療上展示了一定的理論指導意義和潛在的應用價值。
[Abstract]:By trauma, tumor, bone defect caused by infection and disease seriously affect the patient's physical and mental health and quality of life. Autogenous bone and allogenic bone transplantation is still the traditional clinical treatment of bone defect of the common solutions, but these methods have their own limitations limit their application, it is difficult to meet the clinical needs of large therefore, the development of bone repair material has good biological activity has become a research hotspot. Mesoporous silica nanoparticles (mesoporous silica nanoparticles, MSNs) is a kind of nano inorganic particles, has many unique advantages, such as large specific surface area and pore volume, adjustable particle size and pore size and surface modified the characteristics, and has good application prospects in the biomedical field. At present, the researchers began to focus on the application of MSNs in bone tissue engineering. This paper is based on MSNs, according to its surface is easy to change That can encapsulate guest molecules and other characteristics, which can be used to repair bone defect bone repair material and nano porous composite scaffolds, and then study the physicochemical properties and in vivo materials prepared by the osteoinductive ability. The research contents of this paper are summarized as follows: 1. the synthesis of N epsilon n-benzyloxycarbonyl-l-glutamic -L- lysine -N- carboxylic acid anhydride (Lys (Z) -NCA) as monomers, using open ring polymerization reaction in polyethylene imine (PEI) chain formation of poly -L- lysine (PLL), PEI copolymers were prepared by modified PLL (PEI-PLL). Using three different molecular weight PEI (molecular weight 180010000 and 25000) as macroinitiator, three different PEI-PLL copolymers were prepared corresponding (PEI-PLL-1.8k, PEI-PLL-10k and PEI-PLL-25k) by 1H. NMR, FTIR and other characterization techniques confirmed the successful synthesis of three kinds of PEI-PLL copolymers. Then, analyzed three kinds of pei-pll copolymer Buffer capacity, plasmid DNA (pDNA) on pDNA DNaseI binding ability and protection ability. Experimental results show that the cell survival rate, cell toxicity of three kinds of pei-pll copolymers are lower than the molecular weight of 25000 pure Pei (pei-25k). The expression of luciferase, fluorescence microscopy and flow cytometry to study the transfection efficiency of pei-pll copolymer, the results showed that the transfection efficiency of pei-pll-25k was higher than that of the other two kinds of pei-pll copolymer, also than pei-25k showed higher transfection efficiency. Enzyme linked immunosorbent assay (ELISA) experiments show that pei-pll-25k can effectively mediate BMP-2 gene into BMSCs and expression of BMP-2 protein and alkaline phosphatase (ALP) expression and vonKossa staining showed that pei-pll-25k BMP-2 gene transfection mediated by BMSCs can enhance the osteogenic differentiation of BMSCs. Therefore, the molecular weight of Pei was 25000 pei-pll-25k copolymer synthesis at lower cytotoxicity And higher transfection efficiency, can be used as non viral gene vectors for gene therapy of bone related diseases.2.pei-pll-25k copolymer was proved to have high transfection efficiency and low cytotoxicity by edc/nhs chemical method grafting covalently on the surface of MSNs, and further RGD peptide grafted on the surface of the particles, the functionalized MSNs prepared copolymers of pei-pll-25k and RGD modified (msns-ppr). Then, the preparation of msns-ppr by dexamethasone (DEX) and channel load by pdna. cell surface adsorption electrostatic effect of different charge compatibility and blood compatibility results showed that the prepared msns-ppr exhibited good biocompatibility, which is msns-ppr as the foundation. In addition the nano carrier and cell experiments showed that dex@msns-ppr nanoparticles can quickly and effectively by the cellular uptake and distribution in that around the nucleus.Elisa experiment, dex@ msns-ppr could induce The BMP-2 gene into BMSCs and expression of BMP-2 protein. In addition, the activity of ALP, osteogenic related gene expression and alizarin red staining in vitro into bone differentiation experiments, using msns-ppr transfer BMP-2 gene and DEX gene transfer into BMSCs compared to a single BMP-2 and single DEX transfer is more obvious. So the bone induction at the same time, msns-ppr loaded with BMP-2 gene and DEX enhanced osteogenic ability, can be used as a nano bone release system applied in bone tissue engineering by.3. 3- aminopropyl triethoxysilane (APTES) connection, use the edc/nhs chemical method will come from a 73-92 amino acid sequence of BMP-2 protein in graft the surface of the MSNs BMP-2 peptide functionalized MSNs (msns-pep). Then, the DEX load in the pore of MSNs, nano composite BMP-2 polypeptide and DEX osteoblast release system (dex@msns-pep). In vitro cell Show that the viability and cell uptake experiments, BMP-2 peptide functionalized MSNs nanoparticles can improve the biocompatibility and enhanced cellular uptake of nanoparticles. In addition, the experiment in vitro, including the determination of ALP activity, calcium deposition assay and immunohistochemical staining results showed that the prepared msns-pep can promote osteogenic differentiation of BMSCs, and after loading DEX, and further enhance the induced osteogenic differentiation of BMSCs. Ectopic osteogenesis in vivo results showed that in 3 weeks after implantation, the formation of msns-pep and dex@msns-pep can induce new bone. The synergistic effects of BMP-2 polypeptide and DEX, dex@msns-pep than msns-pep induced new bone formation is more obvious. Therefore, dex@msns-pep nano composite BMP-2 polypeptide and DEX system can release osteogenic osteogenic differentiation induced by BMSCs in vitro and in vivo induced ectopic bone formation, has potential application in bone tissue engineering The prospect of.4. in order to simulate organic / inorganic natural bone composition, preparation of functional organic / inorganic scaffolds for bone repair by MSNs and gelatin. First, use MSNs to load the antibiotic vancomycin hydrochloride (Van), and then prepared Van@MSNs., in the preparation process of gelatin scaffold with Van@MSNs nanoparticles prepared by gelatin the composite scaffold composite Van@MSNs (Van@MSNs/Gelatin). Scanning electron microscopy (SEM) images show that with the addition of MSNs increased (5%-20%), the prepared composite scaffold aperture is bigger. At the same time, the addition of MSNs can significantly improve the compressive strength of the composite support, but when the amount of MSNs increased from 5% to 20% the compression strength, but gradually decreased, but still higher than the pure Gelatin scaffold. Show the antibacterial experiment of qualitative and quantitative, Van@MSNs/Gelatin composite scaffold preparation showed good antibacterial effect. In addition, the in vitro cell experiment showed that Van@MSNs/Gelat The adhesion of in composite scaffold can support BMSCs, proliferation and differentiation in vivo. Infected bone defect repair experiments show that the Van@MSNs/Gelatin composite scaffold after 12 weeks of implantation, the defect was repaired well. Therefore, the Van@MSNs/Gelatin composite scaffold preparation can effectively repair materials for infected bone defect. To sum up, the this paper is mainly based on the MSNs vector to construct nano composite double factor osteogenic release system, and further extends to the drug loaded MSNs composite scaffolds, explores the application of these materials in bone repair. This research enriches the application of MSNs in the field of bone tissue engineering, and presents some theoretical guidance and the potential application value in the treatment of bone defect.

【學位授予單位】：東華大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R318.08;R68
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本文編號：1654018