基于聚合物模板的电沉积技术制备仿生骨支架的研究

发布时间：2018-05-17 06:27

本文选题：电沉积技术 + 仿生骨支架　；参考：《东华大学》2015年博士论文

【摘要】：因创伤、肿瘤和疾病等原因造成的骨缺损是临床医学中常见的骨科病症。目前临床上治疗骨缺损最常用的方法有自体骨移植和异体骨移植等,但这些方法均存在各自的局限性(如供体有限、易引发免疫排斥反应等),从而限制了它们在骨修复中的应用,骨组织工程为骨缺损的修复带来了曙光。基于天然骨主要是由胶原纤维、羟基磷灰石(HA)晶体组成的生物矿化体系和具有独特的有机/无机分级结构,理想的仿生骨支架的开发就需要模拟天然骨的主要成分和特殊结构。近年来,电沉积技术在生物医学中的应用日趋广泛,特别是在构建骨支架方面,从金属涂层的制备逐渐向聚合物基底的沉积发展。电沉积能够通过一步法在许多基底材料上沉积磷灰石矿物或其他粒子,与其他技术相比拥有很多优点,如设备简单、易操作,反应条件温和,生产成本低;沉积速度快;沉积物比较均匀等。如果能在聚合物基底上通过电沉积技术沉积骨盐成分,制备可降解的有机/无机复合材料,在结构、成分和功能上仿生天然骨,这将对仿生骨支架的研究具有重要的理论价值和应用前景。基于此,本课题以聚合物为沉积模板,通过电沉积技术制备有机/无机复合仿生骨支架,并对复合支架的理化性能和体内外诱导成骨能力进行研究。具体研究内容主要有:1.首先以聚合物为沉积模板,探究影响电沉积骨盐成分的因素。选取了三种聚合物电纺膜为沉积模板:左旋聚乳酸(plla)、胶原蛋白和壳聚糖;四种金属电极为工作电极:铜(cu)、镍(ni)、钛(ti)和不锈钢(ss),研究了这三种聚合物材料和四种金属电极对沉积磷酸钙盐形貌和结构的影响。结果表明,基底材料能够影响磷酸钙盐的形貌,而其组成差别不大,主要以ha为主。不同的金属电极对磷酸钙盐的形貌和组成均有影响。随后进一步选择plla膜为沉积模板和铜电极为工作电极,研究沉积时间、沉积电压和沉积温度对沉积磷酸钙盐形貌和结构的影响。结果表明,不同的沉积参数使磷酸钙盐晶体呈现不同的形貌和组成,只有在适宜的沉积电压、温度和时间下,才能够生成均一的磷酸钙盐涂层。通过对沉积机理的分析研究,发现涉及的因素主要对磷酸钙盐晶体的成核与生长产生影响。2.进一步研究了电沉积技术对三维支架上磷酸钙盐沉积的影响,并制备有机/无机复合三维支架。采用热致相分离(tips)技术制备具有三维、纳米纤维和多孔结构的plla/聚己内酯(pcl)支架,通过电沉积技术对三维支架进行磷酸钙盐的沉积,获得plla/pcl矿化支架。通过研究沉积电压和时间对支架矿化的影响,得到了纯度较高的ha矿物,且制得的矿化支架能保持多孔结构。为研究矿化支架的体外成骨潜能,将骨髓间充质干细胞(bmscs)种植到支架上并进行培养,发现矿化支架能够促进细胞粘附、提高碱性磷酸酶(alp)活性和促进矿物基质形成。将矿化前后的复合支架植入sd大鼠的颅骨缺损部位12w后,发现矿化支架能够加速颅骨缺损的修复。综上所述,矿化支架从成分上模拟天然骨的无机成分,并结合可降解的聚合物材料,制备的复合支架具有三维多孔纳米纤维结构,能在很大程度上仿生细胞外基质的结构。因此,制备的三维矿化支架很有潜力用于骨组织工程中。3.为探讨添加剂对磷酸钙盐晶体沉积的影响以及对干细胞成骨分化的影响,将锶盐加入到电解液中,探究其对磷酸盐矿化过程的影响。制备plla电纺膜为沉积模板,将不同量的硝酸锶加到电解液中,制备不同浓度的含锶电解液,通过电沉积技术对plla膜进行矿化,采用不同技术研究锶的掺入对沉积的磷酸钙盐晶体的影响。将复合纳米纤维膜与bmscs共培养,研究锶的掺入对细胞增殖和向成骨分化的影响。通过扫描电子显微镜(sem)观察,锶的掺入对沉积物的形貌产生了影响。随着锶浓度的增加,矿物晶体由花簇状变为片状,最后变为球状,且锶的掺入增强了晶体的分散性、使晶型由多晶变为类似单晶的形态。由离子释放行为曲线可以看出,锶的掺入加速了矿物晶体中钙离子和磷酸根离子的释放。细胞实验结果表明,一定浓度锶的掺入能够促进bmscs的增殖,增强alp的活性,提高钙结节的沉积以及骨钙素(ocn)的表达;在一定范围内,掺入锶的含量越高,对细胞的增殖和分化的促进作用越明显。4.进一步研究了电沉积技术在材料表面引入成骨性微载体的可行性,以构建功能性有机/无机骨修复材料。首先合成了氨基化的介孔硅(msns-nh2),用来负载具有成骨潜能的小分子药物地塞米松(dex),制得dex@msns-nh2微载体;采用电沉积技术将其沉积到PLLA/PCL三维支架上,得到DEX@MSNs-NH2/PLLA/PCL复合支架。实验结果表明在沉积电压为3 V时,DEX@MSNs-NH2能够均匀地沉积在PLLA/PCL支架上,获得的复合支架仍能保持三维、多孔和纳米纤维的结构,这将有利于细胞的粘附和向内长入。通过在复合支架上种植BMSCs,研究其对细胞的骨诱导性;并将复合支架植入SD大鼠颅骨缺损处,评估其体内骨修复能力。细胞实验表明复合支架能够支撑BMSCs的粘附、生长和迁移。由ALP活性、茜素红(ARS)染色和骨钙素(OCN)染色结果表明,与PLLA/PCL和MSNs-NH2/PLLA/PCL支架相比,DEX@MSNs-NH2/PLLA/PCL复合支架对BMSCs的成骨分化具有更好的诱导作用。体内颅骨缺损修复实验进一步表明,制得的DEX@MSNs-NH2/PLLA/PCL复合支架能够支撑颅骨缺损处细胞的粘附和迁移,进而加速骨缺损的修复。因此,制备的功能性DEX@MSNs-NH2/PLLA/PCL复合支架有望开发成为有效的骨修复材料。
[Abstract]:Bone defects caused by trauma, tumor and disease are common Department of orthopedics diseases in clinical medicine. The most commonly used methods in clinical treatment of bone defects are autogenous bone graft and allograft bone graft, but these methods have their own limitations (such as limited donor, easy to induce immune rejection, etc.), thus limiting their bone in bone. Bone tissue engineering has brought dawn to the repair of bone defects. Natural bone is mainly composed of collagen fiber, hydroxyapatite (HA) crystal and unique organic / inorganic hierarchical structure. The development of ideal biomimetic bone scaffold needs to simulate the main components and special structures of natural bone. In recent years, electrodeposition has been widely used in biomedicine, especially in the construction of bone scaffolding, the preparation of metal coating from the metal coating to the deposition of polymer substrate. Electrodeposition can deposit apatite or other particles on many base materials by one step method, which has many advantages compared with other technologies, such as set up. It is simple, easy to operate, mild reaction conditions, low production cost, rapid deposition rate, and more uniform sediment. If the bone and salt composition can be deposited on the polymer substrate by electrodeposition technology, biodegradable organic / inorganic composites are prepared, and the structure, composition and function are biomimetic of natural bone, which will be important for the study of biomimetic bone scaffold. Based on this, this paper uses polymer as a deposition template to prepare organic / inorganic composite biomimetic scaffolds by electrodeposition, and studies the physical and chemical properties of the composite scaffold and the ability to induce osteogenesis in vivo and in vivo. The main contents are as follows: 1. first, the polymer was used as a deposition template to explore the influence of electrodeposition. Three kinds of polymer electrospun membranes were selected as deposition templates: L-polylactic acid (PLLA), collagen and chitosan, and four metal electrodes as working electrodes: copper (Cu), nickel (Ni), titanium (TI) and stainless steel (SS). The effects of the three polymeric materials and four metal electrodes on the morphology and structure of the deposited calcium phosphate were investigated. The results show that the substrate can affect the morphology of calcium phosphate, but the difference is small, mainly ha. Different metal electrodes have influence on the morphology and composition of calcium phosphate. Then, the PLLA film is selected as the deposition template and copper electrode as the working electrode. The deposition time, the deposition voltage and the deposition temperature on the sedimentary phosphoric acid are studied. The effect of calcium salt morphology and structure shows that the different deposition parameters make the calcium phosphate crystals with different morphology and composition. Only the homogeneous calcium phosphate coating can be generated only at the suitable deposition voltage, temperature and time. The main factors involved in the calcium salt crystal are found by the analysis of the deposition mechanism. The effect of nucleation and growth on.2. further studies the effect of electrodeposition on the deposition of calcium phosphate on the three-dimensional scaffold and the preparation of an organic / inorganic composite three-dimensional scaffold. The plla/ polyhexyl (PCL) stents with three-dimensional, nanofibers and porous structures are prepared by thermal phase separation (TIPS). The plla/pcl mineralization scaffold was obtained by the deposition of calcium phosphate. By studying the effect of the deposition voltage and time on the mineralization of the scaffold, the high purity ha mineral was obtained, and the prepared mineralized scaffold could maintain the porous structure. In order to study the osteogenic potential of the scaffold in vitro, the bone marrow mesenchymal stem cells (BMSCs) were planted on the scaffold and cultured. It was found that the mineralized scaffold can promote cell adhesion, increase the activity of alkaline phosphatase (ALP) and promote the formation of mineral matrix. The mineralized scaffold can accelerate the repair of the skull defect after the mineralization of the mineralized scaffold into the skull defect site of SD rats, and the mineralized scaffold simulates the inorganic components of the natural bone from the composition. Combined with degradable polymer materials, the composite scaffolds prepared with a three-dimensional porous nanofiber structure can mimic the structure of the extracellular matrix to a large extent. Therefore, the prepared three-dimensional mineralized scaffold has the potential to use.3. in bone tissue engineering to explore the effect of additive on the deposition of calcium phosphate crystals and to the osteogenesis of stem cells. The influence of the differentiation is added to the electrolyte to explore the influence of the strontium salt on the process of phosphate mineralization. The PLLA electrospun membrane is prepared as a deposition template, and different amounts of strontium nitrate are added into the electrolyte, and different concentrations of strontium electrolyte are prepared. The PLLA membrane is mineralized by electrodeposition, and the incorporation of strontium to the deposition of strontium is studied by different techniques. Effects of calcium phosphate crystals. Co culture of composite nanofibers with BMSCs to study the effect of strontium incorporation on cell proliferation and osteogenic differentiation. Through scanning electron microscopy (SEM), the doping of strontium has an effect on the morphology of the sediments. As the concentration of strontium increases, the mineral crystals change from flower clusters to flakes and eventually become spherical. The doping of strontium enhances the dispersivity of the crystal and makes the crystal form from polycrystalline to single crystal. It can be seen from the ion release behavior curve that the doping of strontium accelerates the release of calcium ions and phosphate ions in the mineral crystals. The experimental results show that a certain concentration of strontium can promote the proliferation of BMSCs and enhance the activity of ALP. To improve the deposition of calcium nodules and the expression of osteocalcin (OCN), the higher the content of strontium is, the more obvious the promoting effect on the proliferation and differentiation of the cells, the more.4. further studies the feasibility of the introduction of the osteogenic microcarrier on the surface of the material by electrodeposition technology, in order to construct functional organic / inorganic bone repair materials. Aminated mesoporous silicon (msns-nh2) is used to load the small molecular drug dexamethasone (DEX) with osteogenic potential to produce dex@msns-nh2 microcarrier. The DEX@MSNs-NH2/PLLA/PCL composite scaffold was deposited on the PLLA/PCL three-dimensional stent by electrodeposition technology. The experimental results show that DEX@MSNs-NH2 can sink evenly when the deposition voltage is 3 V. The composite scaffolds retained on the PLLA/PCL scaffold can still maintain three-dimensional, porous and nanofiber structures, which will benefit the adhesion and inward growth of the cells. By planting BMSCs on the composite scaffold, the bone inducibility of the cells is studied, and the composite scaffold is implanted in the skull defect of SD rats, and the bone repair ability in the body is evaluated. The results showed that the composite scaffold could support the adhesion, growth and migration of BMSCs. The results of ALP activity, alizarin red (ARS) staining and Osteocalcin (OCN) staining showed that the DEX@MSNs-NH2/PLLA/PCL composite scaffold had a better induction of osteogenic differentiation of BMSCs compared with PLLA/PCL and MSNs-NH2/PLLA/PCL stents. In vivo skull defect repair experiment entered into one. The results show that the prepared DEX@MSNs-NH2/PLLA/PCL composite scaffold can support the adhesion and migration of cells in the skull defect and accelerate the repair of bone defects. Therefore, the functional DEX@MSNs-NH2/PLLA/PCL composite scaffold is expected to be an effective bone repair material.
【学位授予单位】：东华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08;R687

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