【作者】 张威；

【导师】 毛靖；

【作者基本信息】 华中科技大学 ， 口腔医学， 2014， 博士

【摘要】 1.研究背景与目的仿生再矿化技术利用聚酸类聚电解质稳定的无定形磷酸钙纳米前体可纤维内再矿化牙本质-树脂粘接混合层中胶原稀疏含水丰富区域的裸露胶原,以提高牙本质-树脂粘接层的耐久性能。为了将这一理论转化为临床可用的再矿化策略,可预见的是聚酸类聚电解质稳定的无定形磷酸钙作为可直接利用的终产物相比将再矿化物组分中的钙,磷来源以及仿生类似物分别作为添加剂的方法更加行之有效。本研究阶段检验了氨基功能修饰化的介孔硅纳米颗粒作为无定形磷酸钙纳米前体的转运工具的使用效能,选择性区域电子衍射法证明了被吸附进入到介孔硅纳米颗粒微小间隔中的无定形磷酸钙纳米前体仍保持无定形非晶特性,利用分光光度法测定来源于无定形磷酸钙前体中的钙与磷酸根离子的累积释放浓度,介孔硅颗粒在酸性,中性以及碱性条件下释放无定形磷酸钙前体的动力学机制得到初步探讨,通过选择重组装的Ⅰ型胶原纤维作为矿化模板,介孔硅纳米颗粒作为无定形磷酸钙的纳米缓释工具其纤维内再矿化工型胶原纤维的能力得到进一步的检验。2.材料与方法(1)溶胶法预先合成介孔硅纳米颗粒,正硅酸乙酯(tetraethyl orthosilicate, TEOS)作为硅的来源,氨水作为碱性催化剂,阳离子表面活性剂(cetyltrimethyl ammonium bromide, CTAB)作为结构导向剂,水作为溶剂,在Stober方法的基础上通过精确控制TEOS:NH40H:CTAB:H20四者的摩尔比例1：9.14：0.128：3969达到控制颗粒大小的目的,等到硅酸水解脱水缩合形成二氧化硅框架后表面活性剂通过高温煅烧的方法以去除暴露其中的内孔结构。去除模板剂之后的介孔硅纳米颗粒通过透射电镜(Transimission Electron Microscopy, TEM),热重分析法(Thermogravimetrie Analysis,TGA), X射线衍射法(Powder X-Ray Diffraction,XRD),X射线光电子能谱分析法(X-ray Photoelectron Spectroscopy,XPS),衰减全反射傅里叶变换红外光谱分析法(Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy, ATR-FTIR), zeta电位以及29硅固态核磁共振波谱技术(Solid-state Nuclear Magnetic Resonance, SSNMR)等理化手段对其进行表征。由于聚酸类聚电解质稳定的无定形磷酸钙与介孔硅纳米颗粒表面同带负电荷,去除模板剂的介孔硅纳米颗粒经过后期接枝氨基功能基团变为表面带正电荷的氨基功能修饰化的介孔硅以利于吸附并储存无定形磷酸钙。氨基功能修饰化的介孔硅纳米颗粒通过透射电镜(TEM),扫描透射电镜结合X射线能谱元素分布(分析)(Scanning Electron Transmission equipped with Dispersive Energy Dispersive X-ray Analysis, STEM-EDX),原子力显微镜(Atomic force microscopy),热重分析法(TGA),衰减全反射傅里叶变换红外光谱分析法(ATR-FTIR),29硅固态核磁共振波谱技术(29Si CP-MAS NMR), X射线光电子能谱分析法(XPS),以及氮气吸附脱附实验(N2sorption/desorption)等理化方法进行表征。(2)聚丙烯酸稳定的无定形磷酸钙通过PILP方法在过饱和电解质溶液中预先合成。液体样的无定形磷酸钙渗透吸附到氨基功能修饰化的介孔硅内孔结构中或者是锚定在颗粒的外表面,负载有无定形磷酸钙的的氨基功能修饰化的介孔硅纳米颗粒在TEM, STEM-EDX, AFM等理化手段下进行表征。通过选择性区域电子衍射法(Selected area electron diffraction, SAED),无定形磷酸钙的非晶特性得到检验。(3)每毫克去除模板剂的介孔硅颗粒与氨基功能修饰化后的介孔硅颗粒中的钙,磷以及硅的元素含量通过电感耦合等离子体发射光谱法(Inductive Coupled Plasma-Atomic Emission Spectroscopy, ICP-AES)进行定量分析。通过TEM,无定形磷酸钙在酸性,中性,碱性条件下从氨基功能修饰化的介孔硅颗粒中释放之后的形貌特征得以观察,酸性以及中性条件下的无定形磷酸钙的累积释放浓度通过分光光度法进行确定。(4)利用2-D胶原矿化模型,负载有无定形磷酸钙前体的氨基功能修饰化介孔硅纳米颗粒再矿化Ⅰ型胶原纤维的能力得到检验。含碳透射电镜镍网上的牛皮来源的工型胶原溶液在氨气的中和下自主装成胶原纤维,胶原纤维在1-乙基-3(3-二甲丙氨基)碳化亚胺(1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, EDC)的交联作用下分子结构变得更为稳定。在胶原矿化的前期研究中我们发现直接利用负载无定形磷酸钙前体的氨基功能修饰化介孔硅纳米颗粒在酸性pH=2条件下容易粘附到胶原纤维的外表面,影响对胶原纤维微观结构的观察,因此间接法中介孔硅纳米颗粒先被分散到pH=2的酸性中5天以充分释放无定形磷酸钙,混合液在离心后沉淀绝大部分的介孔硅纳米颗粒,允许无定形磷酸钙存留在上清液中,然后含有胶原纤维的透射电镜镍网漂浮在含有无定形磷酸钙的上清液中37。C存放1-4天。3.结果溶胶法合成的窄粒径分布的介孔硅纳米颗粒平均直径约为50nm,具有规则的六边形孔状结构。聚丙烯酸稳定的无定形磷酸钙与氨基功能修饰化的介孔硅纳米颗粒zeta电位分别为-33.8eV和17.5eV。29Si固态核磁共振与X射线光电子能谱分析法证实氨基成功接枝到介孔硅表面。通过氮气吸附/脱附实验,计算得到介孔硅具有高的表面积953m2/g,孔容量为0.44cm3/g,平均孔径为2.1nm。扫描透射电镜能谱分析以及原子力显微镜表明无定形磷酸钙已经成功的吸附到介孔硅的介孔内与颗粒的外表面。在透射电镜下初步观察了介孔硅颗粒在酸性,中性,碱性条件下释放无定形磷酸钙初始成核粒子的行为,在酸性与碱性条件下大量的初始成核粒子得到释放,相比在碱性条件下以介孔硅的结构破坏作为释放代价,介孔硅在酸性pH=2的条件下既能大量释放无定形磷酸钙,又能维持介孔硅载体的外在框架。通过间接法,无定形磷酸钙在酸性pH=2的条件下大量释放无定形磷酸钙并通过离心沉淀了绝大部分的二氧化硅颗粒,在透射电镜下可观察到矿化前体能渗透进入胶原内的间隔中并随着时间的变化最终转变为沿着胶原长轴纵向分布的磷灰石晶体。更多还原

【Abstract】 Objectives:Biomimetic remineralization improves the longevity of resin-dentin bonds by intrafibrillar mineralization of denuded collagen within resin-sparse, water-rich regions of hybrid layers with polyacid-stabilized amorphous calcium phosphate (Pa-ACP). For clinical translation of this technology, direct delivery of Pa-ACP (end-product) is more effective than delivery of remineralization components (Ca, P sources and biomimetic analogs). This work examined the use of amine-functionalized mesoporous nanosilica as delivery devices for Pa-ACP prenucleation clusters, the stability of the latter that were encapsulated in the nanoconfinements of mesoporous silica was examined by SAED (selected area electron diffraction), the release kinetics of the Pa-ACP loaded mesoporous silica were investigated under acidic, neutral and basic solution by spectrophotometric method, the potential use of this control led-r el ease delivery system for intrafibrillar mineralization of collagen was further examined by choosing reconstitued type-I collagen as mineralization model.Materials and methods:Part-Ⅰ. Mesoporous silica nanoparticles (MSNs) was firstly synthesized using sol-gel process, tetraethyl orthosilicate (TEOS) as Si source, ammonium as catalyst, a kind of cationic surfactant (cetyltrimethyl ammonium bromide, CTAB) was used as structure-directing agents, the size of the particles were controlled according to Stober method by modified the TEOS:NH40H:CTAB:H2Omolar ratio as1:9.14:0.128:3969; the surfactant was removed by calcination after nanosilica formation. MSNs were characterized with TEM (Transimission Electron Microscopy), TGA (thermogravimetric analysis), XRD (Powder X-Ray Diffraction), XPS (X-ray Photoelectron Spectroscopy), ATR-FTIR (Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy), zeta potential and29Si CP-MAS NMR (Solid-State Nuclear Magnetic Resonance Spectroscopy) Because both of Pa-ACP and MSNs have negative zeta-potentials, MSNs were post-functionalized with aminosilane that formed into amine-functionalized MSNs (AF-MSNs) with net positive charge for storage of Pa-ACP prenucleation clusters. AF-MSNs were characterized with TEM, STEM-EDX (Scanning Transmission Electron Microscopy-Energy Dispersive X-ray Analysis), AFM (Atomic force microscopy), TGA, ATR-FTIR,29Si CP-MAS NMR, XPS and N2sorption/desorption (specific surface area, pore size and volume).Part-Ⅱ Polyacylic acid stablized amorphous calcium phosphate (Pa-ACP) were generated in supersaturation solution using PILP method. After Pa-ACP prenucleation clusters were diffused into the inner pores of AF-MSNs or immobilized onto the external surface of the silica nanoparticles, the Pa-ACP prenucleation cluster-loaded AF-MSNs were then characterized with TEM, STEM-EDX, AFM. The amorphous phase of Pa-ACP was characterized by SAED.Part-Ⅲ The total amount of Ca, P and Si in per mg of MSNs and AF-MSNs powder were quantified using ICP-AES (Inductive Coupled Plasma-Atomic Emission Spectroscopy). Morphology of Pa-ACP that released from AF-MSNs at acidic, neutral and alkaline pH was examined using TEM. The release kinetics of Pa-ACP conducted under above conditions were investigated by spectrophotometric method.Part-IV The use of Pa-ACP loaded AF-MSNs to mineralize collagen fibrils was examined using a2-D collagen mineralization model. Bovine skin-derived type I collagen solution was allowed to self-assemble into fibrillar collagen on nickel TEM grids by interacting with ammonia vapor. Reconstituted collagen fibrils were cross-linked with0.3M carbodiimide prior to the mineralization experiments. Pilot work showed that direct application of Pa-ACP loaded AF-MSNs dispersed in acidified deionized water (pH2) to the TEM grids resulted in AF-MSNs adhering to the fibrils’surface, which made it impossible for ultrastructural examination of intrafibrillar mineralization. Thus, an indirect application method was used in which Pa-ACP loaded AF-MSNs were first dispersed in acidified deionized water (pH2) for5days to release Pa-ACPs; centrifuging was performed for4min at4000rpm to precipitate the majority of AF-MSNs, while allowing Pa-ACPs to remain in the solution. Mineralization of collagen fibrils was performed by floating TEM grids over droplets of Pa-ACP containing supernatant at37°C for1-4days.Results:MSNs had a mean diameter of50nm and hexagonal pore arrangement. Zeta-potentials of Pa-ACP and AF-MSNs were-33.8eV and17.5eV, respectively. Amine-functionalization was confirmed using29Si CP-MAS NMR and XPS. AF-MSN had specific surface area of953m2/g, pore volume of0.44cm3/g and mean pore width of2.1nm. STEM-EDX and AFM showed that Pa-ACPs were loaded both within the mesopores and on the external surface of AF-MSNs. Release of Pa-ACP prenucleation clusters from AF-MSN was observed in acidic, neutral and alkaline medium, intensive release of the prenucleation clusters were found in acidic and alkaline conditions, compared to the dissolution of silica framework under pH10, ordered mesostructures of silica nanoparticles remained under pH2. With the sustain release of Pa-ACP from AF-MSNs under pH2and effective elimination of silica nanoparticles, the mineralization precursors could diffuse into the inner compartments of collagen and ultimately formed into apatite crystallites arranged along the fibrils’longitudinal axis.更多还原