Department of Materials and Engineering, Imperial College London, London, UK.
Department of Materials Science and Metallurgy, University of Cambridge, Cambridge, UK.
Acta Biomater. 2015 Jul;20:129-139. doi: 10.1016/j.actbio.2015.03.039. Epub 2015 Apr 4.
To devise new strategies to treat bone disease in an ageing society, a more detailed characterisation of the process by which bone mineralises is needed. In vitro studies have suggested that carbonated mineral might be a precursor for deposition of bone apatite. Increased carbonate content in bone may also have significant implications in altering the mechanical properties, for example in diseased bone. However, information about the chemistry and coordination environment of bone mineral, and their spatial distribution within healthy and diseased tissues, is lacking. Spatially resolved analytical transmission electron microscopy is the only method available to probe this information at the length scale of the collagen fibrils in bone. In this study, scanning transmission electron microscopy combined with electron energy-loss spectroscopy (STEM-EELS) was used to differentiate between calcium-containing biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite). A carbon K-edge peak at 290 eV is a direct marker of the presence of carbonate. We found that the oxygen K-edge structure changed most significantly between minerals allowing discrimination between calcium phosphates and calcium carbonates. The presence of carbonate in carbonated HA (CHA) was confirmed by the formation of peak at 533 eV in the oxygen K-edge. These observations were confirmed by simulations using density functional theory. Finally, we show that this method can be utilised to map carbonate from the crystallites in bone. We propose that our calibration library of EELS spectra could be extended to provide spatially resolved information about the coordination environment within bioceramic implants to stimulate the development of structural biomaterials.
为了制定新的策略来治疗老龄化社会中的骨骼疾病，需要更详细地描述骨骼矿化的过程。体外研究表明，碳酸化矿物可能是骨磷灰石沉积的前体。骨骼中碳酸盐含量的增加也可能对改变机械性能产生重大影响，例如在患病骨骼中。然而，关于骨骼矿物质的化学和配位环境及其在健康和患病组织内的空间分布的信息尚不清楚。空间分辨分析透射电子显微镜是唯一可用于探测骨胶原纤维长度尺度上此类信息的方法。在这项研究中，扫描透射电子显微镜结合电子能量损失光谱（STEM-EELS）用于区分含钙生物矿物质（羟磷灰石、碳酸羟磷灰石、β-磷酸三钙和方解石）。290eV 的碳 K 边峰是碳酸盐存在的直接标志物。我们发现，矿物之间的氧 K 边结构变化最大，允许区分钙磷酸盐和碳酸钙。在碳酸化 HA（CHA）中碳酸盐的存在通过氧 K 边的 533eV 处形成的峰得到证实。这些观察结果通过使用密度泛函理论的模拟得到了证实。最后，我们表明可以利用这种方法来绘制骨骼中结晶物质的碳酸盐图。我们提出，我们的电子能量损失光谱校准库可以扩展，以提供有关生物陶瓷植入物内配位环境的空间分辨信息，从而刺激结构生物材料的发展。
