复相电纺支架缓释rhCEMP1用于牙骨质再生的研究
发布时间：2018-09-18 14:04
【摘要】：牙周病是一种影响广泛的高发病,主要损伤牙齿支持组织,最终导致牙齿的松动脱落。理想的牙周再生,应同时包括牙骨质、牙周膜和牙槽骨的再生。牙骨质的再生是牙周组织的重要组成部分,是目前的牙周组织复合体恢复研究中的热点和难点。在牙周组织再生时,如果缺乏牙周膜和／或牙骨质再生,新生的牙槽骨将占据牙周膜间隙与形成直接结合,从而引起骨粘连。这种病理性、缺乏弹性的结合方式会导致牙齿支持功能的缺失,最终引起牙根吸收。牙骨质再生是牙骨质-牙周膜界面结构再生的先决条件,因此必须采用特异性生长因子诱导牙骨质再生。工程化牙周组织就是在这个条件基础上实现的牙周组织重构。前期研究表明牙骨质蛋白1是一种牙骨质所特有的非胶原蛋白,从组织或真核细胞中提取的人牙骨质蛋白具有促进矿化的作用。因此我们猜想CEMP1正是诱导牙骨质再生最理想的特异性生长因子。牙骨质蛋白1自身的昂贵价格局限了其研究及应用。在蛋白表达中最基本的两种系统分别是原核表达系统和真核表达系统。原核表达使用时间最早,也是目前研究最清楚、最经济实惠的表达系统,同时也是美国FDA批准通过的基因工程表达系统。本实验通过大肠杆菌表达系统得到可与CEMP1抗体特异性结合的rhCEMP1,分子量为34kDa。此蛋白诱导人牙周膜细胞后可使其CEMP1 mRNA表达量升高。实验证明了大肠杆菌原核表达和Ni柱纯化可以获得浓度及纯度较高并具有生物活性的CEMP1蛋白。本研究在仿生生物矿化体系观察浓度范围为0-100 μg/ml的CEMP1对HA晶体48小时生长过程的调控作用。结果发现CEMP影响HA的晶体成核与生长,当CEMP浓度较低(0-50 μg/ml)时,只有短小束状HA晶体生成,矿化效果较差。当CEMP浓度提高到100μg/ml时,大部分晶体的形态变为规整的长针状结构。FTIR显示HA的特征峰1106 cm-1,557cm-1和598 cm-1存在。因此确定CEMP1在体外可以通过提高局部浓度诱导排列整齐的HA晶体形成。为了解决rhCEMP1在体内应用时难以缓慢长效释放的难题,结合牙骨质基质的特点,选用ACP缓释载药系统合成rhCEMP1/ACP纳米缓释颗粒。研究采用PEG稳定的方法获得ACP,并在低温湿化学反应中加载rhCEMP1因子,使得磷酸钙与细胞因子形成稳定的结合。同时通过调节载药量比例,得到了载药量分别为1,2,5,8%的rhCEMP1/ACP纳米缓释颗粒。分析四种rhCEMP1/ACP纳米缓释颗粒的载药量和包封率的关系以及释放谱,数据表明主要有三种释放方式,首日爆释、第一周慢型释放以及一周后的后期快型释放。因此可以针对牙骨质再生的实际需要选用合适的释放谱型的rhCEMP1/ACP纳米缓释颗粒。本研究用静电纺丝的方法获得了具有多孔纳米结构,表面粗糙度高,亲水性良好的复相ACP/PCL/COL支架。本实验发现rhCEMP1/ACP/PCL/COL支架在体外与PDLC共同培养7天,可抑制PDLC增殖；促进成牙骨质细胞分化因子CAP和CEMP1表达,抑制成骨细胞分化因子OCN和OPN表达；将rhCEMP1/ACP/PCL/COL支架在大鼠颅骨标准缺损模型培养4周可诱导类牙骨质组织形成。综上所述,本研究证实了CEMP1在调节HA晶体生长的过程中存在着剂量效应,确定了CEMP1体内牙骨质诱导再生作用。为CEMP1的作用及机制提供了思路,为rhCEMP1在临床中应用于促牙骨质再生提供的研究基础。
[Abstract]:Periodontal disease is a widespread and high-incidence disease, which mainly damages the supporting tissues of the teeth and eventually leads to the loosening of the teeth. The ideal periodontal regeneration should include the regeneration of cementum, periodontal ligament and alveolar bone. In periodontal tissue regeneration, if periodontal ligament and/or cementum regeneration are absent, the new alveolar bone will occupy the periodontal ligament space and form a direct bond, resulting in bone adhesion. This pathological, inelastic bond will lead to the loss of tooth support function and eventually cause root resorption. Cementum regeneration is cementum. The engineered periodontal tissue is based on this condition to achieve periodontal tissue remodeling. Previous studies have shown that cementin-1 is a non-collagen protein unique to cementum, extracted from tissue or eukaryotic cells. Cementoprotein 1 is an ideal specific growth factor for inducing cementum regeneration. The high price of cementoprotein 1 limits its research and application. The two basic systems in protein expression are prokaryotic expression system and eukaryotic expression system. It is the earliest and most economical expression system that has been studied and approved by FDA in the United States. In this study, rhCEMP1, which can specifically bind to CEMP1 antibody, was obtained by E. coli expression system, and its molecular weight is 34 kDa. The results showed that CEMP could regulate the 48-hour growth of HA crystals in a biomimetic mineralization system. CEMP could affect the crystal growth of HA. When the concentration of CEMP increased to 100 ug/ml, the morphology of most of the crystals changed into regular long needle-like structure. FTIR showed that the characteristic peaks of HA were 1106 cm-1,557 cm-1 and 598 cm-1. Therefore, CEMP1 could be enhanced in vitro. In order to solve the problem of slow and long-term release of rhCEMP1 in vivo, combined with the characteristics of cementum matrix, rhCEMP1/ACP nanoparticles were synthesized by ACP sustained-release drug delivery system. ACP was obtained by PEG stabilization method and loaded with rhCEMP1 in low-temperature wet chemical reaction. RhCEMP1/ACP nanoparticles with 1,2,5,8% drug loading were obtained by adjusting the drug loading ratio. The relationship between drug loading and encapsulation efficiency and release spectra of four kinds of rhCEMP1/ACP nanoparticles were analyzed. In this study, the composite ACP/PCL/COL scaffolds with porous nanostructures, high surface roughness and good hydrophilicity were prepared by electrospinning. It was found that rhCEMP1/ACP/PCL/COL scaffold could inhibit the proliferation of PDLC in vitro for 7 days, promote the expression of CAP and CEMP1, and inhibit the expression of OCN and OPN. RhCEMP1/ACP/PCL/COL scaffold could induce the morphology of cementoid tissue after 4 weeks of culture in rat standard skull defect model. In conclusion, this study confirmed the dose-effect of CEMP1 in regulating the growth of HA crystals, and confirmed the role of CEMP1 in inducing cementum regeneration in vivo.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R783.1
本文编号：2248146
[Abstract]:Periodontal disease is a widespread and high-incidence disease, which mainly damages the supporting tissues of the teeth and eventually leads to the loosening of the teeth. The ideal periodontal regeneration should include the regeneration of cementum, periodontal ligament and alveolar bone. In periodontal tissue regeneration, if periodontal ligament and/or cementum regeneration are absent, the new alveolar bone will occupy the periodontal ligament space and form a direct bond, resulting in bone adhesion. This pathological, inelastic bond will lead to the loss of tooth support function and eventually cause root resorption. Cementum regeneration is cementum. The engineered periodontal tissue is based on this condition to achieve periodontal tissue remodeling. Previous studies have shown that cementin-1 is a non-collagen protein unique to cementum, extracted from tissue or eukaryotic cells. Cementoprotein 1 is an ideal specific growth factor for inducing cementum regeneration. The high price of cementoprotein 1 limits its research and application. The two basic systems in protein expression are prokaryotic expression system and eukaryotic expression system. It is the earliest and most economical expression system that has been studied and approved by FDA in the United States. In this study, rhCEMP1, which can specifically bind to CEMP1 antibody, was obtained by E. coli expression system, and its molecular weight is 34 kDa. The results showed that CEMP could regulate the 48-hour growth of HA crystals in a biomimetic mineralization system. CEMP could affect the crystal growth of HA. When the concentration of CEMP increased to 100 ug/ml, the morphology of most of the crystals changed into regular long needle-like structure. FTIR showed that the characteristic peaks of HA were 1106 cm-1,557 cm-1 and 598 cm-1. Therefore, CEMP1 could be enhanced in vitro. In order to solve the problem of slow and long-term release of rhCEMP1 in vivo, combined with the characteristics of cementum matrix, rhCEMP1/ACP nanoparticles were synthesized by ACP sustained-release drug delivery system. ACP was obtained by PEG stabilization method and loaded with rhCEMP1 in low-temperature wet chemical reaction. RhCEMP1/ACP nanoparticles with 1,2,5,8% drug loading were obtained by adjusting the drug loading ratio. The relationship between drug loading and encapsulation efficiency and release spectra of four kinds of rhCEMP1/ACP nanoparticles were analyzed. In this study, the composite ACP/PCL/COL scaffolds with porous nanostructures, high surface roughness and good hydrophilicity were prepared by electrospinning. It was found that rhCEMP1/ACP/PCL/COL scaffold could inhibit the proliferation of PDLC in vitro for 7 days, promote the expression of CAP and CEMP1, and inhibit the expression of OCN and OPN. RhCEMP1/ACP/PCL/COL scaffold could induce the morphology of cementoid tissue after 4 weeks of culture in rat standard skull defect model. In conclusion, this study confirmed the dose-effect of CEMP1 in regulating the growth of HA crystals, and confirmed the role of CEMP1 in inducing cementum regeneration in vivo.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R783.1
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