羧甲基壳聚糖基复合材料的制备及在骨缺损修复应用方面的研究

发布时间：2018-11-19 17:34

【摘要】：羧甲基壳聚糖(CMCS)是壳聚糖的羧甲基化产物,可溶于水,具有良好的生物相容性、生物降解性、pH敏感特性以及可以螯合Ca2+具有成骨活性等特点,已经广泛的应用于组织修复领域。新型骨缺损修复材料是近几年来国内外研究的热点之一,本论文以CMCS为基础制备了几种复合材料,并研究了相关材料用于骨组织缺损修复的可行性。主要研究内容如下:首先,利用CMCS具有良好水溶性且易成膜的特点,制备了CMCS与丝素蛋白(SF)的复合膜,并采用生物相容性良好的京尼平对其进行交联。结果表明:复合膜中SF发生了晶型转化,由silk I结晶结构转化为silk II结晶结构。复合膜表现出良好的亲水性,并有利于小鼠骨髓间充质干细胞的黏附和增殖。其次,利用CMCS中羧基可以鳌合Ca2+的特点,在水热反应中通过CMCS调控羟基磷灰石(HA)的沉积,制备了CMCS/HA复合材料。探讨了CMCS浓度和反应温度对HA颗粒形貌的影响,结果显示,随着CMCS浓度的增加HA由棒状向片状结构转变,而温度对其形貌的影响较小。热分析结果显示当反应溶液中CMCS浓度为0.1%、0.2%、0.4%、0.6%时,所获得的复合材料中有机物CMCS的含量分别为15%、20%、25%、25%。复合材料具有良好的亲水性及生物相容性,细胞实验表明该材料有利于骨髓间充质干细胞的黏附和增殖。为了模拟细胞外基质的纳米纤维形态,采用静电纺丝技术制备了CMCS纳米纤维支架。基于CMCS良好的水溶性,以水为溶剂进行纺丝可避免使用有机溶剂带来的潜在生物安全性问题。通过加入聚氧乙烯(PEO)降低溶液的表面张力,并详细研究了纺丝工艺电压、PEO分子量对纳米纤维形态的影响,结果表明PEO分子量为100万、电压为25kV时可以得到形态良好的纳米纤维。为进一步改善纳米纤维的成骨活性,在5倍模拟体液(5×SBF)中进行仿生矿化制备了CMCS/磷酸钙(CaP)复合纳米纤维。细胞学实验研究表明矿化前后纳米纤维均有利于细胞的黏附和增殖,而矿化的纳米纤维可促进细胞碱性磷酸酶的表达。RT-PCR检测显示矿化的纳米纤维与未矿化纳米纤维相比,可促进主要的成骨标识物(ALP、Runx-2、OCN和COL)的基因表达,更有利于小鼠骨髓间充质干细胞向成骨细胞分化。以大鼠的颅骨缺损模型来评价矿化前后纳米纤维的骨修复能力,micro-CT、苏木精-伊红染色法(HE染色)和马松三色染色(masson三色)结果表明植入矿化纳米纤维4周后,缺损部位尤其是中间部分可以看到有新生骨生成,而空白组和未矿化纳米纤维组新生骨主要是从缺损部位的边缘处向中心长入,12周后矿化纤维组骨缺损部位基本全被新生骨覆盖,而空白组和未矿化纳米纤维组的骨缺损仍未愈合。最后,鉴于CMCS纳米纤维作为骨组织工程支架时必须解决其抗水溶性问题,本论文首次采用反应性电纺技术,通过氧化海藻酸(ADA)的醛基和CMCS的氨基发生席夫碱反应,在静电纺丝CMCS纳米纤维的同时实现ADA对CMCS纤维的原位交联。该技术可望避免传统上采用戊二醛交联剂存在的生物毒性较大的弊端。详细探讨了混合溶液的流变行为及PEO分子量、电压等工艺参数对纳米纤维形貌的影响规律,结果表明在PEO分子量为100万,电压为25kV时可以得到形态良好的CMCS-ADA-PEO复合纳米纤维,通过去离子水浸泡的方法可有效去除PEO获得了CMCS-ADA纳米纤维。经过酸碱溶液及PBS中性溶液浸泡实验证实了该复合纳米纤维支架具有良好的抗水溶性。细胞实验结果表明ADA的加入不仅改善了纳米纤维的抗水溶性,而且CMCS-ADA纳米纤维相比戊二醛交联的CMCS纤维以及单纯海藻酸纳米纤维更有利于细胞的黏附和增殖,以及向成骨细胞分化的能力。
[Abstract]:Methyl chitosan (CMCS) is a methylating product of chitosan, can be dissolved in water, has the characteristics of good biocompatibility, biodegradability, pH sensitivity, and can be used for binding Ca2 + to have bone activity and the like, and has been widely used in the field of tissue repair. The new bone defect repair material is one of the hot spots in recent years, and several kinds of composite materials have been prepared on the basis of CMCS, and the feasibility of the related materials for the repair of bone tissue defects is also studied. The main contents of the study are as follows: First, the CMCS has the characteristics of good water solubility and easy to form, and the composite membrane of CMCS and silk fibroin (SF) is prepared, and the cross-linking of CMCS and silk fibroin (SF) is carried out. The results show that the SF in the composite membrane has a crystalline transformation, and the silk I crystal structure is transformed into a silk II crystal structure. The composite membrane has good hydrophilicity and is beneficial to the adhesion and proliferation of bone marrow mesenchymal stem cells in mice. The CMCS/ HA composite was prepared by controlling the deposition of hydroxyapatite (HA) by CMCS in hydrothermal reaction. The effect of CCS concentration and reaction temperature on the morphology of HA particles was discussed. The results showed that, with the increase of CMCS, the change of the rod-like structure was observed, and the effect of temperature on the morphology of HA was small. The results of the thermal analysis show that the CMCS content in the composite is 15%, 20%, 25% and 25%, respectively, when the CCS concentration in the reaction solution is 0.1%, 0.2%, 0.4%, 0.6%, respectively. The composite material has good hydrophilicity and biocompatibility, and the cell experiment shows that the material is beneficial to the adhesion and proliferation of bone marrow mesenchymal stem cells. In order to simulate the nano-fiber morphology of the extracellular matrix, the CMCS nanofiber scaffold was prepared by electrostatic spinning. The water-soluble, water-based solvent based on CMCS avoids the potential biological safety problems caused by the use of organic solvents. The surface tension of the solution is reduced by the addition of polyethylene oxide (PEO), and the effect of the spinning process voltage and the PEO molecular weight on the morphology of the nano-fiber is studied in detail. The results show that the PEO molecular weight is 1 million and the voltage is 25kV, and the nano-fiber with good morphology can be obtained. In order to further improve the bone-forming activity of the nanofibers, the CMCS/ calcium phosphate (CaP) composite nanofibers were prepared by biomimetic mineralization in 5-fold simulated body fluid (5-SBF). Cytological experiments show that the nano-fibers before and after mineralization are beneficial to the adhesion and proliferation of the cells, and the mineralized nano-fibers can promote the expression of alkaline phosphatase in the cells. The expression of bone markers (ALP, Runx-2, OCN and COL) can be promoted by RT-PCR, which is more beneficial to the differentiation of bone marrow mesenchymal stem cells into osteoblasts. The bone repair ability, micro-CT, hematoxylin-eosin staining (HE staining) and Masson three-color staining (masson three-color) of the pre-and post-mineralized nano-fibers were evaluated by the skull defect model of the rat, and the results showed that after 4 weeks of implantation of the mineralized nano-fibers, in particular in that middle part of the defect part, a new bone formation is seen, and the new bone of the blank group and the unmineralized nano-fiber group is mainly from the edge of the defect part to the center, and the bone defect part of the mineralized fiber group after 12 weeks is completely covered by the newborn bone, The bone defects in the blank and unmineralized nano-fiber groups were still not healed. In the end, in view of the need to solve the water-solubility problem of CMCS nano-fiber as the scaffold of bone tissue engineering, the reactive electrospinning technology is adopted for the first time, and the amino-generating Schiff base of the aldehyde group and the CMCS of the Alginic acid (ADA) is reacted by the reactive electrospinning technology. and the in-situ cross-linking of the ADA to the CMCS fiber is realized at the same time of the electrostatic spinning CMCS nano-fiber. The technology is expected to avoid the disadvantage that the traditional glutaraldehyde cross-linking agent has a large biological toxicity. The influence of the rheological behavior of the mixed solution and the process parameters such as the molecular weight and the voltage of the PEO on the morphology of the nano-fiber is discussed in detail. The results show that the CMCS-ADA-PEO composite nano-fiber with good morphology can be obtained when the molecular weight of PEO is 1 million and the voltage is 25kV. The CMCS-ADA nano-fiber can be effectively removed by the method of soaking in deionized water. The solution of acid-base solution and the neutral solution of PBS confirmed that the composite nano-fiber scaffold had good water-solubility. The results of the cell experiment show that the addition of ADA not only improves the water-solubility of the nano-fiber, but also the CMCS-ADA nano-fiber is more beneficial to the adhesion and proliferation of the cells and the ability to differentiate into the osteoblast than the CMCS-ADA nano-fiber compared with the glutaraldehyde cross-linked CMCS fiber and the simple alginic acid nano-fiber.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08

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