New evidence of surface mineralization of collagen fibrils in wild type zebrafish skeleton by AFM and TEM

Zebrafish (Danio rerio) has been generally accepted as a simple model to investigate the vertebrate tissues. In this study, comparative observations to the structural characteristics of the wild type zebrafish skeletal bone before and after decalcification were performed to gain information about surface mineralization in the collagen-mediated mineralization system using Transmission Electron Microscope (TEM) and Atomic Force Microscope (AFM). TEM observations to the decalcified samples showed empty space between the surfaces of the collagen fibrils. However in the TEM micrographs of the sections without staining and decalcification, hydroxyapatite (HA) crystals are observed to deposit on the surfaces, filling in the space and making the mineralized fibrils compactly aligned, with the diameter increasing to more than 150 nm. Consistent evidence was supplied by the AFM observations, which also indicated that the mineralized fibrils become thicker and more compactly aligned. Moreover, it was revealed that the diameters of the mineralized fibrils increase, as mineralization becomes heavier. In highly mineralized areas, they aggregate into thicker fibers. All of these data have provided the first visual evidence supporting the concept of surface mineralization on the collagen fibrils in the zebrafish skeleton system, by which the structural complexity is achieved.