Visualization of the ultrastructural interface of cells with the outer and inner-surface of coral skeletons

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.