磷酸化壳聚糖促进1型胶原仿生矿化影响的研究

发布时间：2018-06-28 19:41

  本文选题：磷酸化壳聚糖 + 1型胶原　； 参考：《浙江大学》2017年硕士论文

【摘要】：背景骨、软组织的损伤和缺失不仅影响患者的咀嚼功能和面部美观,更成为影响人类生命健康的重大威胁。随着人类经济不断发展,口腔颌面部严重的创伤,肿瘤,炎症等疾病日益增多。不仅如此,在牙科修复领域中,由于酸蚀深度与树脂单体渗透深度的不匹配,粘接界面中已脱矿的胶原纤维裸露而树脂未能渗透包裹富水区域形成。缺乏树脂及矿物保护的裸露胶原机械性能差,且极易被基质金属蛋白酶(MatrixMetalProteinases,MMPs)降解,进一步破坏粘接界面影响树脂-牙本质粘接的耐久性。所以,对骨及牙本质等生物硬组织的修复再生成为当前口腔领域研究的重要课题。仿生矿化是一个应用纳米技术理论来模仿生物矿化过程的概念验证策略。无定形相广泛存在于生物矿化过程中,并作为形成无机相的前驱体参与体内硬组织的形成。通过与矿化相关的蛋白调控无定形相的尺寸以及相转变,以更好的渗透到胶原纤维内部空间形成高度复杂的有机-无机复合生物材料,这种矿化策略是一种很有前景的硬组织修复方法,之前的研究工作中,我们在体外模拟与生物矿化相关的非胶原蛋白(non-collagenous proteins,NCP)的功能特点,使用聚丙烯酸(poly acry lic acid,PAA)稳定的亚稳态无定形相(amorphous calcium phosphate,ACP)转化为羟基磷灰石(hydroxyaptite,HAP)并促进脱矿牙本质的仿生再矿化,值得一提的是,再矿化牙本质的结构和显微机械性能与天然牙本质几乎相似。但是,该方法存在耗时过久的问题。因此,探索更完善耗时更短的的矿化体系是亟须解决的问题。壳聚糖,完全或部分脱乙酰形式的几丁质,在自然界中作为几种活生物体的必需支持结构,其独特的大分子结构具有无毒,抗菌和镇痛的优点。此外,它有良好的粘膜粘附性,显示止血性能,广泛用于止血辅料中,并且是酶促可生物降解的。通过化学修饰合成新的生物功能性壳聚糖衍生物已被广泛研究,通过化学修饰既可保持壳聚糖的原始物理化学和生物化学性质,同时新引入的官能团赋予其新的性质。这种天然聚合物复合材料广泛倾向于合成复合生物材料。在近年来兴起的骨组织工程中,壳聚糖及其衍生材料广泛用于支架材料缓释递送生长因子,促进新骨形成。目的:基于磷酸化壳聚糖良好的生物相容性和螯合钙离子等特性,本实验拟探究磷酸化壳聚糖对1型胶原对生物矿化进程的影响。采用透射电镜(Transmission Electron Microscope,TEM)、傅里叶红外光谱(Fourier Transform Infrared,FTIR)表征ACP颗粒的粒径和相变过程以及1型胶原的矿化情况。探索磷酸化壳聚糖促进1型胶原矿化进程的机制及对体系中磷酸钙矿物相变动力学影响。方法:1.1型胶原溶液的配制:将小牛表皮冻干粉溶于0.1mol/L乙酸溶液(pH = 3.0)配制0.2g/L的胶原溶液,4度储存24h备用。2.1型胶原自组装:0.2g/L的胶原溶液置入含碳透射镍网浸泡,置于密闭含1%氨水的容器,中和4h后移去继续培养21h。为稳定胶原结构,用0.5%戊二醛交联1h,制备透射观察的样本60个。3.磷酸化壳聚糖制备:将1g壳聚糖粉末,5g尿素和10mL磷酸加入到40mL二甲基甲酰胺(DMF)中,并将混合物在150℃下在油浴中连续搅拌1小时。冷却至室温后,过滤溶液,沉淀用蒸馏水和无水乙醇充分洗涤沉淀,然后真空干燥将干燥的Pchi溶解在蒸馏水中得到将50mg/ml的溶液备用。4.再矿化液的配制与胶原矿化:配制氯化钙溶液的中含有计算量的tris,以维持反应液的pH约为7.4-7.6。在钙液中添加一定量的PAA溶液,配制一定浓度的磷酸氢二钠溶液。含一定浓度的氯化钙溶液和磷酸氢二钠溶液各25 ml滴定混合后获得矿化液,置于无菌烧杯中,并加入0.2 wt%NaN3溶液以抑制细菌增殖。最终实验组矿化液分别为CaC121OmM,NaHPO4 6mM,PAA350ug/ml。最后在实验组中加入配制好的Pchi溶液,其终浓度为0.2mg/ml。将组装好的含胶原的镍网(n = 60)随机分为两组置于实验组和空白组矿化液中反应按照特定时间点反应后取出。结果:1.1型胶原矿化进程及微观形貌。TEM结果示实验组样本处理36h后开始矿化,72h后矿化完成,密度更高,胶原增粗,与对照组相比明显加速矿化进程。2.红外光谱结果。实验组中Pchi能与胶原反应修饰胶原表面形成功能基团。3.相变过程。FT-IR结果示36h时,实验组与对照组的ACP均发生了相变,实验组中矿化液中的磷酸钙在较短时间内发生相变。空白组矿化液的磷酸钙48小时开始相变。4.矿物形貌。TEM电镜结果示实验组中的无机物颗粒聚集粘附,而对照组中均一分布。结论:磷酸化壳聚糖(PhosphorylatedChitosan,Pchi)能够有效的促进1型胶原的仿生矿化。红外光谱证实了 Pchi能修饰胶原表面,与胶原发生反应。透射电镜观察Pchi作用下1型胶原的矿化进程加快,胶原表面的无机物含量增多,ACP的相变加快,短时间内形成的等级排列的胶原,与天然矿化胶原一致。根据生物矿化相关蛋白的结构特征,本实验在体外模拟了 NCP调控成矿的作用。通过Pchi对胶原的修饰作用和良好的钙离子螯合作用,结合其良好的抗菌性和良好的生物相容性,在短时间内完成脱矿胶原的再矿化,为提高牙本质树脂粘接复合体的耐久性及骨组织工程的构建提供了一个有意义的仿生策略。
[Abstract]:The damage and loss of the background bone and soft tissue not only affect the chewing function and facial beauty of the patients, but also become a major threat to the human life and health. With the continuous development of the human economy, the severe trauma, tumor, and inflammation of the oral and maxillofacial regions are increasing. In the field of dental repair, the depth of acid erosion and the resin are in the field of dental repair. The mismatching of the penetrant depth of the monomer, the demineralized collagen fibers exposed in the bonding interface and the resin failed to permeate the water rich region. The lack of mechanical properties of the exposed collagen, which is not protected by the resin and minerals, is very easy to be reduced by the matrix metalloproteinase (MatrixMetalProteinases, MMPs), which further destroys the influence of the bonding interface on the resin dentin. Therefore, the repair and regeneration of biological hard tissues such as bone and dentin have become an important topic in the field of oral field. Biomimetic mineralization is a conceptual verification strategy using nanotechnology to imitate the process of biomineralization. The amorphous phase is widely used in the process of biological mineralization and is a precursor to the formation of inorganic phases. The body participates in the formation of hard tissues in the body. Through the mineralization related proteins, the size and phase transition of the amorphous phase are regulated to better penetrate into the internal space of the collagen fibers to form highly complex organic inorganic composite biomaterials. This mineralization strategy is a promising method of hard tissue repair. We simulate the functional characteristics of non collagen (non-collagenous proteins, NCP) related to biomineralization in vitro, and use polyacrylic acid (poly acry LIC acid, PAA) stable metastable amorphous phase (amorphous calcium phosphate, ACP) to be transformed into hydroxyapatite and promote the biomimetic remineralization of the demineralized dentine. It is mentioned that the structure and micromechanical properties of remineralized dentin are almost similar to that of natural dentine. However, this method has a long time-consuming problem. Therefore, it is an urgent problem to explore a more time-consuming and shorter mineralizing system. Chitosan, complete or partially deacetyl form of chitin, is used as a few kinds of activities in nature. The necessary support structure of organisms, its unique macromolecular structure has the advantages of non-toxic, antibacterial and analgesic. In addition, it has good mucous adhesion, hemostasis, widely used in hemostasis excipients and biodegradable. The synthesis of new biofunctional chitosan derivatives by chemical modification has been widely studied. Chemical modification can not only maintain the original physicochemical and biochemical properties of chitosan, but also new properties of the newly introduced functional groups. This natural polymer composite is widely used in the synthesis of composite biomaterials. In recent years, chitosan and its derived materials are widely used in scaffold material in bone tissue engineering. Release growth factor to promote the formation of new bone. Objective: Based on the good biocompatibility of phosphorylated chitosan and chelating calcium ion, the effect of phosphorylated chitosan on the biological mineralization process of type 1 collagen was investigated. Transmission electron microscopy (Transmission Electron Microscope, TEM), Fourier infrared spectroscopy (Fourier Transfor) M Infrared, FTIR) characterized the particle size and phase transition of ACP particles as well as the mineralization of type 1 collagen. Explore the mechanism of the phosphorylated chitosan to promote the mineralization process of type 1 collagen and the effect on the kinetics of calcium phosphate mineral transformation in the system. Method: the preparation of type 1.1 collagen solution: calf surface freeze dry powder in 0.1mol/L acetic acid solution (pH = 3). The collagen solution of 0.2g/L was prepared at 4 degrees to store 24h backup.2.1 collagen self assembly: 0.2g/L collagen solution was immersed in a carbon containing transmission nickel net and was placed in a closed container containing 1% ammonia water. After neutralizing 4h, it was removed to continue to cultivate 21h. as a stable collagen structure, and 1H was crosslinked with 0.5% glutaraldehyde to prepare 60.3. phosphorylated chitosan samples for penetration observation: 1g Chitosan powder, 5g urea and 10mL phosphoric acid were added to 40mL two methyl formamide (DMF), and the mixture was stirred for 1 hours in the oil bath at 150 C. After cooling to room temperature, the filter solution was filtered, the precipitate was washed with distilled water and anhydrous ethanol, and then the dry Pchi was dissolved in distilled water to dissolve 50mg/ml in distilled water. Preparation and collagen mineralization of liquid reserve.4. remineralizing solution: the preparation of calcium chloride solution contains a calculated amount of Tris, in order to maintain the pH of the reaction liquid to add a certain amount of PAA solution in the calcium solution to a certain concentration of two sodium hydrogen phosphate solution. After a certain concentration of calcium chloride solution and two sodium hydrogen phosphate solution are mixed with 25 ml titrations, the mixture is titrated by a mixture of 25 ml. The mineralized liquid was placed in the aseptic beaker, and 0.2 wt%NaN3 solution was added to inhibit the bacterial proliferation. Finally, the experimental group was CaC121OmM, NaHPO4 6mM, and PAA350ug/ml. was added to the experimental group at the end of the experimental group. The final concentration was 0.2mg/ml. and the assembled collagen containing nickel net (n = 60) was randomly divided into two groups. The reaction of the mineralized fluid in the group and the blank group was taken out according to the specific time points. Results: the mineralization process and Micromorphology.TEM of type 1.1 collagen showed that the samples of the experimental group began to mineralized after 36h, and the mineralization was completed after 72h, and the density was higher and the collagen was thickened. Compared with the control group, the mineralization process was obviously accelerated by the.2. infrared spectrum. The Pchi energy in the experimental group was Pchi. When the collagen reaction modified the collagen surface to form the functional group.3. phase transition.FT-IR, the results showed that the ACP of the experimental group and the control group were all phase transition. The phase transition of calcium phosphate in the mineralized fluid in the experimental group was in a short time. The phase transformation of calcium phosphate in the blank group began to phase the phase transformation.4. mineral morphology.TEM electron microscope results showed in the experimental group in the experimental group. Conclusion: PhosphorylatedChitosan (Pchi) can effectively promote the biomimetic mineralization of type 1 collagen. Infrared spectra confirm that Pchi can modify collagen surface and react with collagen. Transmission electron microscope observed that the mineralization process of type 1 collagen was accelerated, collagen was accelerated, collagen was observed. The content of the surface inorganic substance increased, the phase transition of ACP accelerated, and the grade of collagen formed in a short time was in accordance with the natural mineralized collagen. According to the structural characteristics of the biomineralized related proteins, this experiment simulated the role of NCP in regulating the mineralization in vitro. The modification of collagen by Pchi and the good calcium ion chelation were combined with it. Good antibacterial and good biocompatibility and complete remineralization of demineralized collagen in a short time provide a meaningful bionic strategy for improving the durability of dentin resin adhesive complex and the construction of bone tissue engineering.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781
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本文编号：2079128