Despite the improvement in bone's molecular, metabolic and live-cell imaging, histological investigation remains most crucial in bone biology for diagnostic and research purposes. Therefore, fixation of bone samples - especially for electron microscopy investigation- is critical to the ultrastructural analysis. Up to date chemical fixation of bone tissue is performed at room temperature resulting in a compromised ultrastructure of bone sample. Thusly, a conclusion of disease caused ultrastructure alteration to normal ones is compromised. In this study we aim at achieving a close-to-native preservation of bone tissue ultrastructure to enhance the understanding of cell-cell and cell-matrix interaction, mineral composition correlation, and three-dimensional organization.Young male rat bone samples (femora and spine) were fixed using two new methods: 1) microwave assisted chemical fixation (MCF); 2) high pressure freezing (HPF) followed by freeze substitution. Furthermore, HPF was used to fix bone marrow aspirate of rat tibia and reaming debris of a 76 years old male patient. Marrow samples were kept in the HPF aluminum platelets during fixation to prevent sample loss.
Large bone samples showed improved ultrastructure and bone matrix preservation using the MCF method compared to conventional fixation. The method enhanced imaging of fibrillar structure organization, Octeocytes, fibroblasts and osteoclasts preservation allowed better identification of their bilayer-membranes and organelles like mitochondria, lysosomes, nuclei and vesicles. Interestingly, unlike conventional protocols fat cells were vastly well preserved. Bone marrow-aspirate as a liquid sample is challenging to fix, however, using HPF achieved excellent preservation shown for the first time. Megakaryocytes exhibited highly homogenous-cytoplasm and were evenly stained. At high magnification of electron microscopy megakaryocytes cytoplasm appeared dens involving regularly-rounded organelles like mitochondria, nuclei and vesicles as well as rough endoplasmic reticulum.