Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Summary

Undecalcified bone histology provides important information for a variety of clinical and research applications. It is technically challenging, particularly with large size specimens. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them.

Abstract

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images¹ and allows for techniques such as fluorochrome assessment and histomorphometry². Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level.

Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

Protocol

1. The tissue is placed in a sealed container containing 10% phosphate buffered formalin solution³. Ensure minimal exposure to light, if fluorochrome analysis is to be performed, as the fluorochromes are light sensitive.
2. Establish which orientation of the tissue will be required.
3. Trim the specimen to size, in the correct orientation, using a band saw. Ensure safety measures are adhered to. The specimen is then placed into an opaque container, the volume of which should be approximately ten times the size of the specimen to achieve adequate fixation. The specimen should remain in the formalin solution a total of between one and two weeks dependi....