抗骨质疏松药物与微量元素对羟基磷灰石前驱体生长与相转变的研究

发布时间：2018-05-20 12:44

本文选题：相转变 + 阿仑磷酸钠　；参考：《浙江大学》2012年硕士论文

【摘要】：磷灰石(Apatite)是人体骨髂的无机矿物质,主要成分是钙磷酸盐和钙碳酸盐,含少量的钠、镁、和氟化物,多数以羟基磷灰石的形式存在。同时,这种矿物质被普遍认为是幼骨发育中由其他磷酸钙盐前驱体转化而来。大量研究表明磷酸八钙(OCP)存在于动物和人体幼骨发育阶段,并且被广泛视为羟基磷灰石(HA)的重要前驱体相,在骨的矿化过程中具有重要作用。研究表明,人工合成的OCP比HA具有更为优良的生物活性和降解性,并在骨损伤修复中显示出优良的骨传导和骨诱导特性。另一方面,中老年人群中的骨质疏松症及骨质疏松骨折并发症问题日益突出,并已成为全球性的重大公共健康问题。目前临床治疗手段主要是服用二膦酸盐类药物和富钙膳食补充剂。二膦酸盐类药物给药方式局限于口服或静脉注射,其生物利用率较低,并伴随有明显的副作用；此外其作用机理也尚不明确,目前的研究大多集中于分子生物学方向,对磷灰石晶体与二膦酸盐的相互作用研究相对不足。而骨代谢过程中必需的一些微量元素对成骨细胞的增殖、分化和矿化具有良好的促进作用,显示出优良的促进骨再生活性,对开发新型用于承载治疗骨科疾病的药物体系等方面具有重要意义。如果我们能进一步了解二磷酸盐、微量元素等对骨矿化初期HA前驱体的生物学效应机制,势必会对促进骨质疏松性骨折愈合人工修复材料的设计和构建带来新的启示。本文以二膦酸盐和微量元素与磷酸八钙晶体的相互作用为研究对象,运用体外矿化体系,系统研究了抗骨质疏松药物阿仑膦酸钠(AS)以及微量元素(锌、锶、镁)等对OCP形核、生长和相转化的基本规律,试图揭示药物小分子或者磷酸钙异质金属离子对OCP纳米晶生长和相转化的剂量关系,为骨质疏松症防治临床用药和新型显著促进骨质疏松性骨损伤再生特定需求修复用材料的研究开发建立理论基础。本文的主要研究结果如下： 1.考虑到骨矿化过程细胞与外基质相互作用特点,本文首先借鉴了细胞膜磷脂双分子层特征,在体外仿生矿化体系建立中,引入临界胶束浓度水平的双亲小分子物质CTAB。初步探索实验表明,CTAB存在与否并不影响矿物质的沉积时间和相转化效率,但是在CTAB存在条件下磷酸钙矿物质纳米晶和其图簇形态形貌更为规整,这极为有利于在显微条件下考察纳米晶形态演化。因此,后续研究AS或微量元素介导磷灰石矿物前躯体成核、生长和相转化研究中均存在该临界胶束浓度水平的双亲小分子物质。 2.阿仑膦酸钠(AS)介导磷灰石矿物质前躯体生长的试验中,我们发现在较短的陈化时间内(无机盐溶液滴加完成1h后),在极低浓度的AS条件下(2～8μM),在OCP成核条件下却优先出现水合磷酸氢钙(Dicalcium Phosphate Dihydrate, DCPD)带状纳米晶,AS越低,这一现象更为显著；当AS浓度达到16μM后,在陈化1h后矿化沉积产物为羟基磷灰石,并且其纳米晶尺度显著降低,形成高度聚集的纳米晶团簇。该实验结果表明,AS在极低溶度下就具备显著调控磷灰石前躯体相转化的作用。 3.基于上述研究基础,我们进一步探讨不同AS浓度水平矿化产物岁陈化时间变化的相转化和形态演化规律。结果发现,在极低AS浓度条件下(2～8μM),DCPD前驱体稳定性逐渐提高,2μM、4μM和8μM AS条件下分别在陈化1、3和8 h候检测到DCPD的存在；类似地,DCPD在转化为OCP后。在上述AS浓度水平,陈化24、72和168 h候检测到该相的存在。同时,尽管DCPD和OCP纳米晶在溶液中发生相转化,但是纳米晶形态却没有显著性变化,表明该过程是原位晶相转化,而不是溶解-再结晶过程。上述研究结果表明,AS这种抗骨质疏松药物小分子在对幼骨矿物熟化和骨代谢中也具有调控作用。 4.在针对无机离子调控磷酸钙纳米晶的实验研究中,Zn离子浓度在相对Ca离子浓度的2～4‰水平时,则显示出对磷酸钙纳米晶的形态和相转化产生促进作用,在较高Sr离子浓度水平(40%),仍然不可见对OCP成核条件下矿化产物的影响。Mg离子作为细胞为基质中的宏量元素,我们的研究也显示该离子在较高水平(10～20%)才会对矿化产物纳米晶的形貌与物相产生影响。这些研究结果表明,不同微量元素对磷灰石前躯体矿化形象存在显著差异,Zn离子的影响最为突出,Sr离子则难以看到介导磷灰石前躯体相变的影响,说明该无机离子具有稳定磷灰石前躯体物质的作用。综上所述,本论文的主要研究结果有助于让我们对具有防治骨质疏松症的二膦酸盐小分子和骨代谢微量元素等物质在直接调控幼骨发育和骨骼代谢中的作用的新认识,这对进一步理解二膦酸盐小分子和骨代谢微量元素的生物学功效以及新型骨质疏松性骨损伤材料的设计和构筑方面具有重要意义,同时为骨质疏松症防治用药剂量、给药方式等提供有益的知识。
[Abstract]:HA is widely regarded as the important precursor phase of hydroxyapatite ( HA ) , and has become a major global public health problem .
In addition , the mechanism of action is not clear . The current researches focus on the molecular biology direction , and the interaction between apatite crystals and diphosphonate is relatively insufficient . Some trace elements necessary for bone metabolism have a good effect on the proliferation , differentiation and mineralization of osteoblasts .

In this paper , based on the interaction of diphosphonate and trace elements with octaccalcium phosphate crystal , the basic rules of anti - osteoporosis drugs ( AS ) and trace elements ( Zn , Sr , Mg ) and other elements ( Zn , Sr , Mg ) and other elements ( Zn , Sr , Mg ) on the growth and phase transformation of anti - osteoporosis drugs were studied .

1 . Considering the characteristics of the interaction between bone mineralization process cells and outer matrix , the double - parent small molecular substance CTAB with critical micelle concentration level was introduced in this paper . The results showed that the presence or absence of CTAB did not affect the deposition time and phase transformation efficiency of the minerals . However , it was very helpful to study the evolution of nano - crystalline morphology under the condition of CTAB .

2 . In the experiment of Alendronate ( AS ) - mediated precursor growth of apatite , we found that under the condition of low concentration of AS ( 2 - 8 渭M ) , the lower the level of AS ( 2 - 8 渭M ) , the lower the level of AS , the lower the AS , the lower the AS , the lower the AS .
When the concentration of AS reached 16渭m , the mineralization deposit was hydroxyapatite after 1 h of aging , and its nano - crystal size decreased significantly , forming a highly concentrated nanocrystalline cluster . The experimental results show that AS has the function of significantly regulating the transformation of apatite precursor phase at very low solubility .

3 . Based on the above - mentioned research foundation , we further discussed the phase transformation and morphology evolution of the aging time of mineralization products with different concentrations of AS . The results showed that DCPD existed at 1 , 3 and 8 h of aging under extremely low AS concentrations ( 2 - 8 渭M ) , while the stability of DCPD precursor was gradually increased .
Similarly , after conversion of DCPD to ocp , the presence of the phase was detected at the above AS concentration levels , aged 24 , 72 and 168 h . At the same time , despite the phase inversion of DCPD and ocp nanocrystals in solution , there was no significant change in the morphology of the nanocrystals , indicating that the process was in - situ crystallization rather than dissolution - recrystallization .

4 . In the experimental study on the regulation of calcium phosphate nanocrystals with inorganic ions , the effect of Zn ion concentration on the morphology and phase transformation of the calcium phosphate nano - crystal is shown . The results show that the influence of different trace elements on the morphology and phase of the mineralization product under the condition of high Sr ion concentration ( 40 % ) is not visible .

In conclusion , the main results of this paper are helpful for us to understand the role of diphosphonate small molecule and bone metabolism trace elements in the control of immature bone development and bone metabolism directly , which is of great significance for further understanding the biological efficacy of the diphosphonate small molecule and trace elements of bone metabolism and the design and construction of new osteoporosis bone injury materials .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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