Manipulating the chemistry and nanotopography of cultured diatoms for the application in tissue regeneration technologies

这一博士学位的总体目标是研究硅藻(微藻)吸收和将选定的金属离子结合到它们的硅质细胞壁(称为锥体)的能力。这是通过一种简单的“体内培养”方法来实现的，在这种方法中，所需金属的盐分被溶解到硅藻的培养介质中。一旦我们确认成功吸收，我们将转移重点，通过改变培养条件来优化最大限度地吸收金属离子的条件。在硅藻结合了金属离子后，将对它们进行表征，特别注意金属吸收后发生的任何化学或物理变化。如上所述，我们将培养我们自己的硅藻，由我们的合作者(圣克劳德州立大学马修·朱利叶斯博士)提供，并改变多种条件和参数，如金属和营养物质的浓度，以可用的光照等。一旦硅藻成功生长，将通过一系列分析技术测量它们的金属吸收。金属离子加入到果壳中可能会改变纳米形貌(主要是孔分布和大小)，任何进入有机相的离子都可能改变脂肪酸的组成。利用原子力显微镜、能谱分析、扫描电子显微镜和热重分析等方法对材料的物理/结构特征进行了分析。对于化学/脂肪酸分析，使用的方法包括GCMS和ICP。我们将使用这些技术来监测金属的掺入情况，并尝试优化条件，以允许最大限度地吸收。在这一阶段之后，可以进行溶解研究，以尝试并确定金属离子释放动力学。这项研究之所以特别令人感兴趣，有两个关键原因：首先，这项研究应该阐明硅藻吸收金属并将其结合到其锥体中的过程--先前的工作表明，钙和钛被结合到锥体中，但关于这一过程的细节还不是很清楚。因此，我们希望扩展已知被硅藻利用的金属数据库，并进一步了解这些金属离子对生物化学和球果结构的影响。这项研究令人感兴趣的另一个核心原因是在这项研究将提供的理解基础上可能衍生并进一步发展的生物材料技术的潜在应用。硅藻本身产生非常复杂的多孔纳米结构，每一代都可以完美地复制，而且精度是目前的纳米制造过程中难以复制的(这是极其昂贵的，而且使用危险材料)。如果我们能够利用这些生物纳米材料的“工厂”，并开始用替代金属离子掺杂这些管状材料，我们可能会有一条更环保、更便宜的纳米材料途径。然后我们可以通过加入离子来调整这些结构-在我们的研究中，我们选择了特定的金属离子，这些离子在当前的文献中已经被证明对骨细胞有有益的影响，以激发骨再生和增加骨密度(因此，可能是启发新的药物/治疗骨质疏松症等骨骼恶化疾病的初步研究)。到目前为止，没有很多没有引起免疫反应的无麻烦的植入材料，并且其他多孔纳米材料制造困难和昂贵，通常需要危险的化学物质，已经被证明是“硅藻生物二氧化硅是无毒的，不会引发促炎反应”，因此，提供了一种相对良性和廉价的途径来获得高度多孔的可植入纳米结构，希望它们将被证明包含了以某种方式帮助骨愈合/再生的金属离子。

The overall objectives of this PhD are to investigate the ability of diatoms (microalgae) to uptake and incorporate a selection of metal ions into their silica cell walls (known as frustules). This is intended to be achieved via a simple "in-vivo culturing" method, in which the salts of the desired metals are dissolved into the diatoms' culture medium. Once we confirm successful uptake, we will then shift the focus to optimise the conditions for maximum metal ion uptake, through altering the culturing conditions. After the diatoms have incorporated the metal ions they will then be characterised, with particular attention to any chemical or physical changes that occur post metal uptake.As stated above we will culture our own diatoms, provided by our collaborators (Dr Matthew Julius, St Cloud State University), and vary multiple conditions and parameters, such as concentrations of metals and nutrients, to available lighting etc. Once the diatoms have been successfully grown their metal uptake will be measured through a range of analytical techniques. The metal ions that are incorporated into the frustule may change the nanotopography (mainly pore distribution and size), and any ions that are incorporated into the organic phase may change the composition of fatty acids. To analyse the physical/structural features, methods such as AFM, EDX, SEM and TGA will be used. For the chemical/fatty acid analysis, methods used will include GCMS & ICP.We will use these techniques to monitor metal incorporation and try to optimise conditions to allow for maximum uptake. After this stage dissolution studies may be carried out to try and determine the metal ion release kinetics. This research is of particular interest for two key reasons:Firstly, this research should shed light on the process by which diatoms take up and incorporate metals into their frustules - prior work has shown that Ca and Ti are incorporated into frustules, however not a great amount of detail is known about the process. Therefore, we wish to extend the database of metals known to be utilised by diatoms, and further understand what effects these metal ions have on the biochemistry and frustule structure. The other core reason for this research being of interest is the potential applications in biomaterial technology that may be derived and further developed from the understanding this research will provide. Diatoms themselves produce very intricate porous nanostructures that are perfectly replicated each generation, and with precision that would be hard to replicate from current nanofabrication processes (which are extremely costly and utilise hazardous materials). If we can harness these biological nanofabricating "factories" and begin doping these frustules with substitute metal ions we may have a more environmentally friendly & inexpensive pathway to nanomaterials. We can then tune these structures with the ions being incorporated - within our research we have chosen particular metal ions that have been shown within the current literature to have beneficial effects on bone cells to inspire bone regeneration and increase bone density (therefore, may be the preliminary research to inspire new drugs/therapies for bone deterioration diseases like osteoporosis).To now there are not many hassle free implantable materials that do not cause an immune response, and other porous nanomaterials are difficult and expensive to manufacture, typically requiring hazardous chemicals, It has already been shown that "diatom biosilica is non-toxic and does not invoke a pro-inflammatory response", therefore offering a relatively benign and inexpensive pathway to highly porous implantable nanostructures, and hopefully they will be shown to incorporate metal ions that in one way or another help bone healing / regeneration.