Despite the improvement in bones 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. In this study, we aim at achieving a close-to-native preservation of bone tissue ultrastructure to enhance the understanding of cellcell and cellmatrix interaction, mineral composition correlation, and three-dimensional organization.
Young male rat bone samples (femora and spine) were fixed using new methods; microwave-assisted chemical fixation (MCF), and 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-year-old male patient.
Large bone samples showed improved ultrastructure and bone matrix preservation using the MCF method compared to conventional fixation. The method enhanced preservation of fibrillar structure organization, octeocytes, fibroblasts and osteoclasts and 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. MCF has shortened the process of bone samples preparation for TEM investigation and allowed better fixatives penetration improving ultrastructure preservation. The processing enhanced the fixation steps such as fixative-crosslinking, dehydration, infiltration of embedding medium and polymerization. Interestingly, HPF processed samples exhibited a close to genuine ultrastructure preservation of bone marrow as a unique sample type.
14 May 2016 - 17 May 2016