Purpose: Cracks in bone and articular calcified cartilage (ACC) of the subchondral plate heal by intercalation of a highly mineralised infill material which disappears with demineralisation. This material may also extruded into hyaline articular cartilage (HAC) as High Density Mineralised Protrusions (HDMPs), which may fragment and act as a cutting and grinding agent, damaging HAC from within. We wish to know more about the structure and composition of HDMPs. We have prepared very thin sections from the front face of bone blocks embedded in PMMA - which had previously been studied by backscattered electron scanning electron microscopy (BSE-SEM) and x-ray microtomography (XMT) - by the new technique of laser-ablation machining and wanted to know if this method could be applied to this rather intractable problem.

Methods: Samples are subject to DESS MRI and XMT before cutting slabs for PMMA embedding. The PMMA blocks are further reduced to include regions having HDMPs, prepared by polishing for backscattered electron imaging in a scanning electron microscope (BSE SEM) and higher resolution XMT. Now the block surface in the XMT reconstruction corresponds exactly with that seen by BSE SEM. The block face is stuck to a glass slide using cyano-acrylate adhesive and placed on the ‘Tissue Surgeon’ equipment (LLS Rowiak LaserLabSolutions GmbH, Germany). This works by scanning a femtosecond pulsed 1030 nm laser through a cutting plane in the specimen. Laser energy is focussed by a high numerical aperture objective lens into a very small specimen volume and for an extremely short period, generating a high instantaneous laser flux that obliterates the specimen only at the focal spot. The focussed, pulsed laser beam is scanned along a 1mm line, and this swathe is translated in an x-y snake scan to cover the entire area of the block, which is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on 4000 grit silicon carbide polishing paper is used to remove 1-2 μm of cutting relief from the surface of the section. The block face and the underlying few microns comprise the section after laser cutting. We examine the uncoated section on the slide in the SEM for BSE imaging. For light microscopy, we apply a coverslip using glycerol as an easily removable mounting medium which does not attack PMMA or cyanoacrylate., and can go backwards and forwards to LM methods. Polarised light microscopy is best done at this stage, before staining. Many stains work with the embedding resin left in place. Images are married using homemade software.

Results: With the new method we produce and use thinner sections (e.g., in the 6 to 10µm range rather than the 50-100µm range in classical ground sections). We get these sections – undeformed, intact, undecalcified - from PMMA block surfaces, so that we may have perfect correlation with x-ray microtomography (XMT, µCT), confocal LM of the block before cutting, BSE-SEM for mineral content studies, iodine stained BSE-SEM for soft tissue histology (cells, osteoid, cartilage, ligament, tendon, periosteum etc) and with all LM methods, including staining, phase, polarised light etc.

Conclusions: Laser ablation microtomy produces high quality, thin sections of both hard, mineralised and dense fibrous connective tissues of any sort – even of single thin trabeculae – which can be studied with any light microscopic method as well as BSE-SEM. One sample can be studied in both the SEM and the LM. SEM becomes an extension of the LM range of methods. We are able to perform serial sectioning of the extraordinarily-difficult-to-handle HDMP features which are usually lost, down the drain, with the decalcifying solutions rampant in run-of-the-mill histopathological laboratories.

HDMP = high density mineralised protrusion. HAC = hyaline articular cartilage. ACC = articular calcified cartilage.