Jeger Rina, Lichtenfeld Yona, Peretz Hagit, Shany Boaz, Vago Razi, Baranes Danny
Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
J Electron Microsc (Tokyo). 2009 Apr;58(2):47-53. doi: 10.1093/jmicro/dfp005. Epub 2009 Feb 13.
Crystalline, porous biomaterials, such as marine invertebrate skeletons, have been widely used for functional reconstruction of human tissues like bone and dental implants. Since in such an abrasive microenvironment adequate cell-material interactions are crucial for a successful treatment, it is of great importance to improve the means to examine these interactions. We developed a method that reveals the ultrastructure of the interface between coral skeletons and cultured neural cells to a higher quality than do traditional methods as it does not include damaging procedures like decalcification or sectioning non-decalcified skeletons. It is rather based on generating two electron opacity distinct Araldite masks, of the skeleton and its surrounding, by polymerizing them to different durations. The contrast created at the border of the two masks outlined the fine and fragile crystals of the coral skeleton's outer and inner surfaces and their contact sites with the cells. The skeleton's internal structure contains a mesh of narrow (few microns wide) and large channel-shaped gaps interrupted by irregular-shaped crystalline material. Neural cells grew on the skeleton surface by stretching between crystal tips, with occasional rearrangements of cytoskeletal fibers located near the anchorage focal adherence points. Cell processes infiltrated the skeleton interior by stretching between inter-surface crystals and by adjusting their volume to the space of the conduits they grew into. The technique advances the study of coral biology and of neural cells-hard biomaterial interaction; it can be applied to other biomaterials and cell types and open new ways for studying tissue development and engineering.
晶体状多孔生物材料，如海洋无脊椎动物的骨骼，已被广泛用于人体组织的功能重建，如骨和牙种植体。由于在这种有磨损性的微环境中，充分的细胞与材料间的相互作用对于成功治疗至关重要，因此改进检测这些相互作用的方法非常重要。我们开发了一种方法，该方法能以比传统方法更高的质量揭示珊瑚骨骼与培养的神经细胞之间界面的超微结构，因为它不包括脱钙或切割未脱钙骨骼等破坏性程序。它是基于通过将骨骼及其周围环境聚合不同时长来生成两个电子不透明度不同的环氧树脂掩膜。在两个掩膜边界处产生的对比度勾勒出了珊瑚骨骼外表面和内表面的精细且脆弱的晶体及其与细胞的接触部位。骨骼的内部结构包含由不规则形状的晶体材料中断的狭窄（几微米宽）和大通道状间隙的网格。神经细胞通过在晶体尖端之间伸展在骨骼表面生长，在锚定粘着斑附近偶尔会重新排列细胞骨架纤维。细胞突起通过在表面间晶体之间伸展并根据它们生长进入的管道空间调整其体积而渗透到骨骼内部。该技术推动了珊瑚生物学以及神经细胞与硬生物材料相互作用的研究；它可应用于其他生物材料和细胞类型，并为研究组织发育和工程开辟新途径。
