介孔硅纳米成骨释放系统的构筑及骨修复应用

发布时间：2018-03-23 15:18

  本文选题：介孔硅　切入点：功能化　出处：《东华大学》2016年博士论文

【摘要】：由外伤、肿瘤、感染和疾病等因素造成的骨缺损严重影响患者的身心健康和生活质量。传统的自体骨和异体骨移植仍然是临床上治疗骨缺损的常用方案,但由于这些方法存在自身的局限性限制了它们的广泛应用,难以满足庞大的临床需求,因此开发具有优良生物活性的骨修复材料成为研究热点。介孔硅纳米粒子(mesoporous silica nanoparticles,MSNs)是一种纳米级的无机粒子,具有许多独特的优点,如大的比表面积和孔体积、可调的粒径和孔径以及易修饰的表面特性等,在生物医学领域具有较好的应用前景。当前,研究者们开始聚焦MSNs在骨组织工程的应用。本课题以MSNs为基体,根据其表面易改性、可封装客体分子等特点,构建可用于骨缺损修复的纳米骨修复材料以及多孔复合支架材料,然后对所制备的材料的理化性能和体内外成骨诱导能力进行研究。本文的研究内容概括为以下几个部分:1.以合成的Nε-苄氧羰基-L-赖氨酸-N-羧酸酐(Lys(Z)-NCA)为单体,利用开环聚合反应在聚乙烯亚胺(PEI)链上生成聚-L-赖氨酸(PLL),制备PLL修饰的PEI共聚物(PEI-PLL)。使用了三种不同分子量的PEI(分子量为1800,10000和25000)作为大分子引发剂,制备了对应的三种不同PEI-PLL共聚物(PEI-PLL-1.8k,PEI-PLL-10k和PEI-PLL-25k)。通过1H NMR、FTIR等表征技术证实三种PEI-PLL共聚物的成功合成。然后,分析了三种pei-pll共聚物的缓冲能力、质粒dna(pdna)结合能力和对pdna的dnasei保护能力。细胞成活率实验结果显示,三种pei-pll共聚物的细胞毒性都低于分子量为25000的纯pei(pei-25k)。通过荧光素酶表达、荧光显微镜观察和流式细胞术研究pei-pll共聚物的转染效率,结果显示pei-pll-25k的转染效率高于其他两种pei-pll共聚物,同时也比pei-25k表现更高的转染效率。酶联免疫吸附测定(elisa)实验表明,pei-pll-25k能够有效介导bmp-2基因进入bmscs并表达bmp-2蛋白。而且,碱性磷酸酶(alp)表达和vonkossa染色结果显示,pei-pll-25k介导bmp-2基因转染bmscs,能够增强bmscs的成骨分化。因此,以pei分子量为25000合成的pei-pll-25k共聚物表现更低的细胞毒性和更高的转染效率,可作为非病毒基因载体用于骨相关疾病的基因治疗。2.pei-pll-25k共聚物被证明具有高转染效率和低细胞毒性,通过edc/nhs化学法将其共价接枝在msns表面,并进一步将rgd多肽接枝在粒子表面,制得pei-pll-25k共聚物和rgd修饰的功能化msns(msns-ppr)。然后,制备的msns-ppr能够通过孔道负载地塞米松(dex)并且可以通过表面异种电荷的静电作用吸附pdna。细胞相容性和血液相容性结果显示,制备的msns-ppr表现出良好的生物相容性,这为msns-ppr作为纳米载体奠定了基础。另外,细胞实验表明,dex@msns-ppr纳米粒子能快速、有效被细胞摄取并分布在细胞核周围。elisa实验证实,dex@msns-ppr能够介导bmp-2基因进入bmscs并成功表达bmp-2蛋白。此外,由alp活性、成骨相关基因表达和茜素红染色的体外成骨分化实验显示,利用msns-ppr共传递bmp-2基因和dex进入bmscs相比单一bmp-2基因传递和单一dex传递表现更加明显的成骨诱导作用。因此,同时负载bmp-2基因和dex的msns-ppr表现增强的成骨诱导能力,可作为纳米成骨释放系统应用于骨组织工程。3.通过3-氨基丙基-三乙氧基硅烷(aptes)的连接作用,利用edc/nhs化学法将来源于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)。接着,将dex负载在msns的孔道内,制备复合bmp-2多肽和dex的纳米成骨释放系统(dex@msns-pep)。体外细胞成活率和细胞摄取实验表明,bmp-2多肽功能化的msns能提高纳米粒子的细胞相容性并增强细胞对纳米粒子的摄取。另外,体外诱导实验,包括alp活性测定、钙沉积分析和免疫组化染色,结果证明制备的msns-pep能够促进bmscs的成骨分化,而且负载dex后,进一步增强对bmscs成骨分化的诱导作用。体内异位成骨实验结果显示,在植入后的3周,msns-pep和dex@msns-pep都能诱导新骨的形成。在bmp-2多肽和dex的协同作用下,dex@msns-pep比msns-pep诱导更加明显的新骨形成。因此,复合bmp-2多肽和dex的dex@msns-pep纳米成骨释放系统能够在体外诱导bmscs的成骨分化以及在体内诱导异位成骨,在骨组织工程中具有潜在的应用前景。4.为了模拟天然骨组织的有机/无机组成成分,利用msns和明胶制备功能性有机/无机骨修复支架。首先,用MSNs来负载抗生素盐酸万古霉素(Van),制得Van@MSNs。然后,在明胶支架的制备过程中加入Van@MSNs纳米粒,制备出复合Van@MSNs的明胶复合支架(Van@MSNs/Gelatin)。扫描电镜(SEM)图片显示,随着MSNs的加入量增多(5%-20%),制得的复合支架的孔径就越大。同时,MSNs的加入能明显提高复合支架的压缩强度,但是当MSNs的加入量从5%提高到20%,其压缩强度反而逐渐下降,但仍比纯Gelatin支架好。定性和定量的抑菌实验显示,制备的Van@MSNs/Gelatin复合支架表现良好的抑菌效果。另外,体外细胞实验表明Van@MSNs/Gelatin复合支架能够很好地支持BMSCs的粘附、增殖和分化。体内感染性骨缺损修复实验表明,在Van@MSNs/Gelatin复合支架植入12周后,缺损部位得到良好的修复。因此,制备的Van@MSNs/Gelatin复合支架可有效作为感染性骨缺损的修复材料。综上,本论文主要以MSNs为载体构建了复合双因子的纳米成骨释放系统,并进一步将载药MSNs延伸到复合支架,探究了这些材料在骨修复中的应用。本文的研究工作丰富了MSNs在骨组织工程领域的应用,并在骨缺损的治疗上展示了一定的理论指导意义和潜在的应用价值。
[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.

【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R68
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本文编号：1654018