skelet.AL

Skelet.AL specialises in morphological analysis, using micro-computed tomography (micro-CT) both ex-vivo and in-vivo; is a fully equipped modern histology facility for the preparation and analysis (histomorphometry) of both decalcified and undecalcified bone specimens; and has recently acquired the TissueFaxs 200, a slide scanner able to scan up to 200 slides of immunostained tissue sections (brightfield and fluorescence), along with its flow cytometry like analysis software package.

In recent years, skelet.AL has expended its range of expertise through collaborations with specialised laboratories such as the Bone Biochemistry Lab and the INSIGNEO Integrative Musculoskeletal Biomechanics Group.

More information is available on the skelet.AL website.

Histology

The bone analysis lab is a fully equipped and modern histology laboratory, containing all the instrumentation necessary for the preparation and analysis of soft tissues and both decalcified and undecalcified bone specimens. The techniques available within the lab include:

• Fixation and decalcification of hard tissue.
• Processing and embedding of specimens into paraffin wax and resin media.
• Preparation of thin tissue sections from paraffin wax embedded samples.
• Preparation of thin tissue sections from resin-embedded, undecalcified tissue samples using a heavy-duty, automated Leica RM2265 microtome and a tungsten carbide or diamond knife.
• A variety of staining techniques to demonstrate both bone structure and cellular components within the tissue sample are available. These include standard techniques such as haematoxylin and eosin, toludine blue and more specific stains such as Von Kossa to demonstrate mineralised tissue and tartrate-resistant acid phosphatase (TRAP) staining to identify osteoclast cells.
• Histomorphometric analysis of bone samples using ‘state of the art’ image analysis systems (see histomorphometry page for more information).

Morphological Analysis of Bone

The preparation of sections from bone samples can give valuable information on bone structure but the process may also lead to damage of the fine structures within the bone. Computerised tomography (CT) can be used to give non-invasive quantitative, 3-dimensional detailed information on bone. The bone analysis laboratory uses a Skyscan 1172 desktop x-ray high-resolution microtomograph, which provides the following applications:

• Non-invasive analysis of small bone samples (max size 68mm x 75mm)
• 10 Megapixel camera enables acquisition of high-resolution images (pixel size < 2µm).
• Quantitative analysis of parameters such as trabecular number, trabecular area/volume and bone porosity can be obtained using bone analysis software.
• 3D models of the sample or selected areas of the sample can be reconstructed from the CT scans.
• Measurement of mineral content within bone samples and the bone density can be assessed
• No pre-treatment of the bones is required and the bone samples can be processed histologically once the scanning has been completed.

Histomorphometry

Histomorphometric analysis can provide a great deal of information of the cellular structure and components of bone. In the bone analysis laboratory thin tissue sections (stained with appropriate histological dyes) are analysed using the Osteomeasure histomorphometry system (OsteoMetrics, Inc). The system can calculate over 175 parameters of bone including trabecular area and length, number of osteoclasts and number of osteoblasts in accordance with the ASBMR Standard Nomenclature.

The rate of new bone formation may also be measured using histomorphometry. Fluorescent labels (such as tetracycline, calcein and xylenol orange) can be used which bind to newly formed bone at the bone/osteoid (non-mineralised bone) interface.